Proc Natl Acad Sci U S A . 2005 Jan 11; {Epub ahead of print} Thermodynamic prediction of protein neutrality; Bloom JD et al.; We present a simple theory that uses thermodynamic parameters to predict the probability that a protein retains the wild-type structure after one or more random amino acid substitutions . Our theory predicts that for large numbers of substitutions the probability that a protein retains its structure will decline exponentially with the number of substitutions, with the severity of this decline determined by properties of the structure . Our theory also predicts that a protein can gain extra robustness to the first few substitutions by increasing its thermodynamic stability . We validate our theory with simulations on lattice protein models and by showing that it quantitatively predicts previously published experimental measurements on subtilisin and our own measurements on variants of TEM1 beta-lactamase . Our work unifies observations about the clustering of functional proteins in sequence space, and provides a basis for interpreting the response of proteins to substitutions in protein engineering applications. Genomics Proteomics Bioinformatics, 2003 Aug, 1(3), 173 - 9 Integration of G-protein coupled receptor signaling pathways for activation of a transcription factor (EGR-3); Tan X et al.; We recently reported the use of a gene-trapping approach to isolate cell clones in which a reporter gene had integrated into genes modulated by T-cell activation . We have now tested a panel of clones from that report and identified the one that responds to a variety of G-protein coupled receptors (GPCR) . The beta-lactamase tagged EGR-3 Jurkat cell was used to dissect specific GPCR signaling in vivo . Three GPCRs were studied, including the chemokine receptor CXCR4 (Gi-coupled) that was endogenously expressed, the platelet activation factor (PAF) receptor (Gq-coupled), and beta2 adrenergic receptor (Gs-coupled) that was both stably transfected . Agonists for each receptor activated transcription of the beta-lactamase tagged EGR-3 gene . Induction of EGR-3 through CXCR4 was blocked by pertussis toxin and PD58059, a specific inhibitor of MEK (MAPK/ERK kinase) . Neither of these inhibitors blocked isoproterenol or PAF-mediated activation of EGR-3 . Conversely, beta2- and PAF-mediated EGR-3 activation was blocked by the p38, specific inhibitor SB580 . In addition, both beta2- and PAF-mediated EGR-3 activation could be synergistically activated by CXCR4 activation . This combined result indicates that EGR-3 can be activated through distinct signal transduction pathways by different GPCRs and that signals can be integrated and amplified to efficiently tune the level of activation. Medicina (B Aires), 2004, 64(2), 113 - 9 {Survey of chest physicians regarding COPD diagnosis and treatment}; Sivori ML et al.; A survey on COPD diagnostic procedures, treatment and management was conducted in a group of 517 chest physicians randomized from a list of the 1121 affiliates to the Asociacion Argentina de Medicina Respiratoria . One hundred eighty-seven responses were obtained (36.2% of the questionnaires mailed) . They treat an average of 53.3 COPD patients every month . Twenty-four percent of them had mild, 41.8% moderate and 33.8% severe disease (GOLD criteria) . Only clinical criteria for diagnosis of COPD, clinical criteria + spirometry (S), and clinical criteria + S + chest X ray were used by 2.9, 23.4 and 73.7% of responders, respectively . Seventy percent of responders believed that chronic asthma without bronchodilator response must be included in the COPD definition . Only 14.1% of responders performed S in every office visit . Cardiac function was assessed using clinical criteria, electrocardiogram and echocardiogram by 90.6, 80.6 and 73.8% of responders, respectively, while 98.3% stated that they trained most of their patients in the inhalation technique . Metered Dose Inhaled was the first option for bronchodilators administration (64.8%) followed by nebulization (16.5%), dry powder inhalation (13.7%) and oral administration (4.8%) . First option for chronic therapy in severe COPD patients was the association of anticholinergic drug (AC) + short acting beta2-agonists (SABA) (65.5%), AC alone (18.8%), long acting beta2-agonists (LABA) (9.7%), inhaled corticosteroids (IC) (3.5%) and SABA alone (2.8%) . Corticosteroids and antibiotics were prescribed in severe COPD exacerbation by 92.5 and 70% of responders, respectively . First choice antibiotic formulation was beta-lactamics + beta-lactamase inhibitors in 39% of the responders followed by fluorquinolones in 23.7%, macrolides in 17.5% and beta-lactamics in 12.5% . Lastly, 12.7% of COPD patients received long-term domiciliary oxygen therapy . 59.3% of them were prescribed pulmonary rehabilitation, 94.1% vaccination against influenza and 91.4% pneumococcal vaccination . Thirty seven percent of the patients continued to smoke . Most of reponses regarding diagnosis and exacerbation treatment were in agreement with recommendations of international guidelines . For maintenance treatment the association of AC + SABA was commonly recommended as first option, whereas IC and LABA were rarely prescribed. Acta Biochim Pol, 2004, 51(4), 925 - 31 Escherichia coli small heat shock proteins IbpA/B enhance activity of enzymes sequestered in inclusion bodies; Kuczynska-Wisnik D et al.; Escherichia coli small heat shock proteins, IbpA/B, function as molecular chaperones and protect misfolded proteins against irreversible aggregation . IbpA/B are induced during overproduction of recombinant proteins and bind to inclusion bodies in E . coli cells . We investigated the effect of DeltaibpA/B mutation on formation of inclusion bodies and biological activity of enzymes sequestered in the aggregates in E . coli cells . Using three different recombinant proteins: Cro-beta-galactosidase, beta-lactamase and rat rHtrA1 we demonstrated that deletion of the ibpA/B operon did not affect the level of produced inclusion bodies . However, in aggregates containing IbpA/B a higher enzymatic activity was detected than in the IbpA/B-deficient inclusion bodies . These results confirm that IbpA/B protect misfolded proteins from inactivation in vivo. Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 57 - 62 Epub 2004 Dec 23. The modular architecture of protein-protein binding interfaces; Reichmann D et al.; Protein-protein interactions are essential for life . Yet, our understanding of the general principles governing binding is not complete . In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules . The boundaries between modules are defined by clustering the contact map of the interface . We show that mutations in one module do not affect residues located in a neighboring module . As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small . To the contrary, within their module, mutations cause complex energetic and structural consequences . Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography . Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface . The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. Antimicrob Agents Chemother, 2005 Jan, 49(1), 358 - 65 Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals; Mulvey MR et al.; A study designed to gain baseline information on strains of Escherichia coli displaying resistance to cefoxitin in Canada is described . A total of 29,323 E . coli isolates were screened at 12 participating hospital sites as part of an extended-spectrum beta-lactamase surveillance initiative . A total of 411 clinically significant, nonrepeat isolates displaying reduced susceptibilities to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000 . Two hundred thirty-two isolates were identified as resistant to cefoxitin . All cefoxitin-resistant strains were subtyped by pulsed-field gel electrophoresis, and of these, 182 strains revealed a unique fingerprint and 1 strain was untypeable . PCR and sequence analysis of the ampC promoter region revealed 51 different promoter or attenuator variants and 14 wild-type promoters . Three promoter regions were interrupted by insertion elements, two contained IS10 elements, and one contained an IS911 variant . PCR and sequence analysis for the detection of acquired AmpC resistance (by the acquisition of ACT-1/MIR-1, CMY-2, or FOX) revealed that 25 strains contained CMY-2, including 7 of the strains found to have wild-type promoters . The considerable genetic variability in both the strain fingerprint and the promoter region suggests that AmpC-type resistance may emerge spontaneously by mutation of sensitive strains rather than by the spread of strains or plasmids in the hospital setting. J Virol, 2005 Jan, 79(2), 918 - 26 Studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha; Yonezawa A et al.; The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates . To date, no effective therapies have been identified . To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope . Fusion was detected by cleavage of the fluorogenic substrate CCF2 by beta-lactamase-Vpr incorporated into virions and released as a result of virion fusion . Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1 . These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion . Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol) . Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion . Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs . In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion . We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells . Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis . Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens. FEMS Microbiol Lett, 2004 Dec 15, 241(2), 229 - 232 Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli; Perilli M et al.; An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase . The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme . The N-terminal amino acid sequence was determined to be NH(2)-QTADVQ.. . Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates . Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme. Biochemistry, 2004 Dec 21, 43(50), 15737 - 45 An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins; Shimizu-Ibuka A et al.; Previous crystallographic structural analysis of extended-spectrum beta-lactamase Toho-1 predicted that the high flexibility of beta-strand B3, the region that contains a conserved KTG motif and forms one wall of the substrate-binding site, could be one of the key features contributing to Toho-1 activity toward third-generation cephalosporins . To investigate whether this possible flexibility really affects the substrate profile of this enzyme, two Toho-1 mutants have been produced, G238C and G238C/G239in, in which the glycine residue at position 238 was replaced with a cysteine and an additional glycine residue was inserted . Our intent was to introduce a disulfide bond between the cysteine residues at positions 69 and 238, and thus to lock the position of beta-strand B3 . The results of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration indicated formation of a new disulfide bridge in the G238C mutant, although disulfide bond formation was not confirmed in the G238C/G239in mutant . Kinetic analysis showed that the activity of the G238C mutant decreased drastically against third-generation cephalosporins, while its catalytic efficiency against penicillins and first-generation cephalosporins was almost identical to that of the wild-type enzyme . This result was consistent with the prediction that flexibility in beta-strand B3 was critical for activity against third-generation cephalosporins in Toho-1 . Furthermore, we have determined the crystal structure of the G238C mutant enzyme to analyze the structural changes in detail . The structural model clearly shows the introduction of a new disulfide bridge and that there is no appreciable difference between the overall structures of the wild-type enzyme and the G238C mutant, although the introduced disulfide bond slightly influenced the positions of Ser237 on beta-strand B3 and Asn170 on the Omega loop . The results of our kinetic and structural analyses suggest that the flexibility of beta-strand B3, as well as the positions of Ser237 and the Omega loop, is critical for the substrate specificity expansion of Toho-1. J Med Chem, 2004 Dec 16, 47(26), 6556 - 68 Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum beta-lactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates; Venkatesan AM et al.; The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A and C serine beta-lactamase inhibitors is described . These compounds proved to be very potent inhibitors of the TEM-1 and AmpC beta-lactamases and less so against the class B metallo-beta-lactamase CcrA . In combination with piperacillin, their in vitro activities enhanced susceptibility of all class C resistant strains from various bacteria . Crystallographic structures of a serine-bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have been established to resolutions of 2.0 and 1.4 A, respectively, and refined to R-factors equal 0.163 and 0.145 . In both beta-lactamases, a seven-membered 1,4-thiazepine ring has formed . The stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has both R and S configurations in the GC1 intermediate . Hydrophobic stacking interactions between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes . The formation of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization. Protein Eng Des Sel . 2004 Dec 1; {Epub ahead of print} Construction of stabilized proteins by combinatorial consensus mutagenesis; Amin N et al.; We constructed stabilized variants of beta-lactamase (BLA) from E . cloacae by combinatorial recruitment of consensus mutations . By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E . cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions . Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability . The stability of these isolates suggest that all tested mutations make additive contributions to protein stability . A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and 8 mutations that destabilized the protein . A second generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization . The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees higher than the starting molecule and contained 8 consensus mutations . Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis . Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability. Phys Med Biol, 1994 Nov, 39(11), 1801 - 9 Dielectric studies of native, unfolded and intermediate forms of beta-lactamase; Bone S; Time-domain dielectric spectroscopy has been applied to native, intermediate and unfolded beta -lactamase structures both in the hydrated solid state and in solution . Gravimetric and dielectric measurements indicate that nearly three times as many water molecules are multiply hydrogen bonded to the unfolded compared with the native protein . By contrast, the primary hydration population of the state A intermediate is only 20% larger than that of the folded enzyme . Analysis of the beta -dispersions in terms of rotational relaxation of the protein's permanent dipole has indicated that there is no significant difference in the dimensions of the three structures . This is not consistent with the findings of previous studies employing different experimental techniques. Chem Biol, 2004 Nov, 11(11), 1483 - 7 A molecular switch created by in vitro recombination of nonhomologous genes; Guntas G et al.; We have created a molecular switch by the in vitro recombination of nonhomologous genes and subjecting the recombined genes to evolutionary pressure . The gene encoding TEM1 beta-lactamase was circularly permuted in a random fashion and subsequently randomly inserted into the gene encoding Escherichia coli maltose binding protein . From this library, a switch (RG13) was identified in which its beta-lactam hydrolysis activity was compromised in the absence of maltose but increased 25-fold in the presence of maltose . Upon removal of maltose, RG13's catalytic activity returned to its premaltose level, illustrating that the switching is reversible . The modularity of RG13 was demonstrated by increasing maltose affinity while preserving switching activity . RG13 gave rise to a novel cellular phenotype, illustrating the potential of molecular switches to rewire the cellular circuitry. Langmuir, 2004 Nov 23, 20(24), 10464 - 73 Immobilization of the enzyme beta-lactamase on biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)-coated sensor chips: a study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing; Zhen G et al.; Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors . In this study, the very efficient enzyme beta-lactamase is used as a model protein . The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme . The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent . beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate . The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface . Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site . Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency . In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics . All tested enzyme variants turned out to be active upon immobilization at the polymeric surface . However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site . No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers. Nucleic Acids Res . 2004 Nov 10;32(20):e158. Combinatorial codon-based amino acid substitutions; Yanez J et al.; Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time . A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase . Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols . This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues . Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides . Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region. Hum Mol Genet, 2005 Jan 1, 14(1), 19 - 38 Epub 2004 Nov 10. The hereditary spastic paraplegia protein spastin interacts with the ESCRT-III complex-associated endosomal protein CHMP1B; Reid E et al.; Pure hereditary spastic paraplegia is characterized by length-dependent degeneration of the distal ends of long axons . Mutations in spastin are the most common cause of the condition . We set out to investigate the function of spastin using a yeast two-hybrid approach to identify interacting proteins . Using full-length spastin as bait, we identified CHMP1B, a protein associated with the ESCRT (endosomal sorting complex required for transport)-III complex, as a binding partner . Several different approaches confirmed the physiological relevance of the interaction in mammalian cells . Epitope-tagged CHMP1B and spastin showed clear cytoplasmic co-localization in Cos-7 and PC12 cells . CHMP1B and spastin interacted specifically in vitro and in vivo in beta-lactamase protein fragment complementation assays, and spastin co-immunoprecipitated with CHMP1B . The interaction was mediated by a region of spastin lying between residues 80 and 196 and containing a microtubule interacting and trafficking domain . Expression of epitope-tagged CHMP1B in mammalian cells prevented the development of the abnormal microtubule phenotype associated with expression of ATPase-defective spastin . These data point to a role for spastin in intracellular membrane traffic events and provide further evidence to support the emerging recognition that defects in intracellular membrane traffic are a significant cause of motor neuron pathology. Biochemistry, 2004 Nov 9, 43(44), 14111 - 7 Inhibitor-resistant class A beta-lactamases: consequences of the Ser130-to-Gly mutation seen in Apo and tazobactam structures of the SHV-1 variant; Sun T et al.; A bacterial response to the clinical use of class A beta-lactamase inhibitors such as tazobactam and clavulanic acid is the expression of variant beta-lactamases with weaker binding affinities for these mechanism-based inhibitors . Some of these inhibitor-resistant variants contain a glycine mutation at Ser130, a conserved active site residue known to be adventitiously involved in the inhibition mechanism . The crystallographic structure of a complex of tazobactam with the Ser130Gly variant of the class A SHV-1 beta-lactamase has been determined to 1.8 A resolution . Two reaction intermediates are observed . The primary intermediate is an acyclic species bound to the reactive Ser70 . It is poorly primed for catalytic hydrolysis because its ester carbonyl group is completely displaced from the enzyme's oxyanion hole . A smaller fraction of the enzyme contains a Ser70-bound aldehyde resulting from hydrolytic loss of the triazoyl-sulfinyl amino acid moiety from the primary species . This first structure of a class A beta-lactamase lacking Ser130, the side chain of which functions in beta-lactam binding and possibly in catalysis, gives crystallographic evidence that the acylation step of beta-lactam turnover can occur without Ser130 . Unexpectedly, the crystal structure of the uncomplexed Ser130Gly enzyme, also determined to 1.8 A resolution, shows that a critical Glu166-activated water molecule is missing from the catalytic site . Comparison of this uncomplexed variant with the wild-type structure reveals that Ser130 is required for orienting the side chain of Ser70 and ensuring the hydrogen bonding of Ser70 to both Lys73 and the catalytic water molecule. Antimicrob Agents Chemother, 2004 Nov, 48(11), 4482 - 4 Pharmacodynamics of ceftazidime plus the serine beta-lactamase inhibitor AM-112 against Escherichia coli containing TEM-1 and CTX-M-1 beta-lactamases; Bowker KE et al.; A strain of Escherichia coli containing TEM-1 and CTX-M-1 was tested in an in vitro pharmacokinetic model against ceftazidime with and without AM-112, a serine beta-lactamase inhibitor . Ceftazidime alone was less effective than ceftazidime plus AM-112, and a single dose was more effective than three fractionated doses. Antimicrob Agents Chemother, 2004 Nov, 48(11), 4217 - 25 OXA-60, a chromosomal, inducible, and imipenem-hydrolyzing class D beta-lactamase from Ralstonia pickettii; Girlich D et al.; A chromosomally encoded oxacillinase, OXA-22, had been characterized from Ralstonia pickettii PIC-1 that did not explain by itself the resistance profile of this strain to beta-lactams . Thus, further analysis of the genetic background of this species led to the identification of another oxacillinase, OXA-60, that was expressed only after beta-lactam induction . This chromosomally encoded oxacillinase shared 19% amino acid identity with OXA-22 . It has a narrow-spectrum hydrolysis profile that includes imipenem . OXA-60-like enzymes were identified in several R . pickettii strains . Gene inactivation and induction studies of the bla(OXA-60) and bla(OXA-22) genes in R . pickettii identified the relative contribution of each oxacillinase to the resistance profile of R . pickettii to beta-lactams. Bioorg Med Chem, 2004 Nov 15, 12(22), 5807 - 17 Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors; Venkatesan AM et al.; Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics . Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases . To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics . However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors . Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria . To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates . The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds. J Biol Chem, 2004 Dec 31, 279(53), 55728 - 36 Epub 2004 Oct 20. Oligomerization of the {gamma}-aminobutyric acid transporter-1 is driven by an interplay of polar and hydrophobic interactions in transmembrane helix II; Korkhov VM et al.; The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers . Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization . In artificial transmembrane segments, interhelical interactions are stabilized by polar residues . We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1) . We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation . Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly . The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking . Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position) . Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e . sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)) . These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments. Anal Biochem, 2004 Nov 15, 334(2), 344 - 55 A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication; Zuck P et al.; This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication . The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells . The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate . Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window . Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors . These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window . HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively. Nucleic Acids Res . 2004 Sep 30;32(17):e136. Protein evolution by codon-based random deletions; Osuna J et al.; A method to delete in-phase codons throughout a defined target region of a gene has been developed . This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode . Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis . The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase . Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found . The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space. Antimicrob Agents Chemother, 2004 Oct, 48(10), 4050 - 3 AmpC beta-lactamase in an Escherichia coli clinical isolate confers resistance to expanded-spectrum cephalosporins; Mammeri H et al.; Cloning, sequencing, and biochemical analysis identified a novel AmpC-type beta-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate . This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E . coli, hydrolyzed ceftazidime and cefepime significantly. Antimicrob Agents Chemother, 2004 Oct, 48(10), 3980 - 8 Antibody mapping of the linear epitopes of CMY-2 and SHV-1 beta-lactamases; Hujer AM et al.; Knowledge of the amino acids that define recognition of anti-beta-lactamase antibodies is critical to the interpretation of sensitivity and specificity of these antibodies when they are used in a clinical or research setting . To this end, we mapped the epitopes of the CMY-2 and SHV-1 beta-lactamases by using the SPOT synthesis method . Eight linear epitopes in SHV-1 and seven linear epitopes in CMY-2 were identified by using anti-SHV-1 and anti-CMY-2 polyclonal antibodies, respectively . The epitopes of SHV-1 were mapped to amino acids at the Ambler positions ABL 28 to 38, 42 to 54, 88 to 100, 102 to 114, 170 to 182, 186 to 194, 202 to 210, and 276 to 288 . In the epitope spanning amino acids 102 to 114, alanine and X-Scan analysis demonstrated that D104, Y105, P107, and S109 are essential residues for antibody recognition . In the epitope containing amino acids 170 to 182, N170, L173, P174, G175, and D176 were immunodominant . In CMY-2 beta-lactamase, amino acids 4 to 16, 70 to 79, 211 to 223, 274 to 286, 289 to 298, 322 to 334, and 343 to 358 of the mature enzyme defined the major linear epitopes . A detailed analysis of the recognition sites that are located in an area analogous to the omega loop of class A beta-lactamases (V211 to V223) showed that the amino acids Q215 to E219 are important in antibody binding . Incubation of CMY-2 beta-lactamase with a 10-fold molar excess of anti-CMY-2 antibody for 60 min resulted in greater than 80% inhibition of nitrocefin hydrolysis . A 10-fold molar excess of anti-SHV-1 antibody reduced the activity of SHV-1 by 69% . Analysis of the CMY-2 and SHV-1 structures suggest that this reduction of hydrolytic activity may be due in part to the direct binding of antibodies to the omega loop, thereby hindering access of substrate to the active site. Virology, 2004 Oct 10, 328(1), 36 - 44 HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion; Cavrois M et al.; We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells . This assay involves the use of virions containing beta-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of beta-lactamase . Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr . Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating . We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells . Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry . Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane . Int J Antimicrob Agents, 2004 Oct, 24(4), 320 - 6 Dissemination of Escherichia coli producing AmpC-type beta-lactamase (CMY-11) in Korea; Kim JY et al.; Among the 51 clinical isolates collected from a university hospital in Korea, nine isolates were resistant to cephamycins . Nine isolates were shown to produce CMY-11 and these also included three isolates producing TEM-1 . The results from ERIC-PCR revealed that dissemination of CMY-11 was due to outbreaks of resistant species and to the intra-species spread of resistance to cephamycins in Korea . CMY-11 beta-lactamase genes from nine clinical isolates that were responsible for resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin and amoxicillin-clavulanic acid, were cloned and characterised . A sequence identical to the common regions in In6, In7 and a novel integron from pSAL-1 was found upstream from bla(CMY-11) gene at nucleotide 1-71 . Eighteen nucleotides between position 71 and 72 were inserted into the bla(CMY-11) gene. Bioorg Med Chem Lett, 2004 Oct 18, 14(20), 5117 - 20 Benzopyranones with retro-amide side chains as (inhibitory) beta-lactamase substrates; Adediran SA et al.; 3-(N-Benzylcarbamoyl)-7-carboxy-3, 4-dihydro-2H-1-benzo-pyran-2-one and its 8-carboxy analogue have been synthesized and evaluated as potential (inhibitory) substrates of beta-lactam-recognizing enzymes . These compounds are bicyclic delta-lactones with retro-amide (with respect to classical beta-lactams) side chains . They were found to be comparably effective as substrates of typical class A, C and D beta-lactamases as analogous benzopyranones bearing 'normal' amide side chains . The new 8-carboxy derivative, however, formed a much more (1000-fold) tightly-bound acyl-enzyme with a class C beta-lactamase than did its 'normal' analogue, and thus provides a structural lead to new inhibitors of this class of beta-lactamase. Antimicrob Agents Chemother, 2004 Sep, 48(9), 3579 - 82 Biochemical characterization of laboratory mutants of extended-spectrum beta-lactamase TEM-60; Caporale B et al.; Three mutants of the extended-spectrum beta-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis . The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background. J Mol Biol, 2004 Aug 27, 341(5), 1295 - 315 Estimating the prevalence of protein sequences adopting functional enzyme folds; Axe DD; Proteins employ a wide variety of folds to perform their biological functions . How are these folds first acquired? An important step toward answering this is to obtain an estimate of the overall prevalence of sequences adopting functional folds . Since tertiary structure is needed for a typical enzyme active site to form, one way to obtain this estimate is to measure the prevalence of sequences supporting a working active site . Although the immense number of sequence combinations makes wholly random sampling unfeasible, two key simplifications may provide a solution . First, given the importance of hydrophobic interactions to protein folding, it seems likely that the sample space can be restricted to sequences carrying the hydropathic signature of a known fold . Second, because folds are stabilized by the cooperative action of many local interactions distributed throughout the structure, the overall problem of fold stabilization may be viewed reasonably as a collection of coupled local problems . This enables the difficulty of the whole problem to be assessed by assessing the difficulty of several smaller problems . Using these simplifications, the difficulty of specifying a working beta-lactamase domain is assessed here . An alignment of homologous domain sequences is used to deduce the pattern of hydropathic constraints along chains that form the domain fold . Starting with a weakly functional sequence carrying this signature, clusters of ten side-chains within the fold are replaced randomly, within the boundaries of the signature, and tested for function . The prevalence of low-level function in four such experiments indicates that roughly one in 10(64) signature-consistent sequences forms a working domain . Combined with the estimated prevalence of plausible hydropathic patterns (for any fold) and of relevant folds for particular functions, this implies the overall prevalence of sequences performing a specific function by any domain-sized fold may be as low as 1 in 10(77), adding to the body of evidence that functional folds require highly extraordinary sequences. Acta Crystallogr D Biol Crystallogr, 1995 Sep, 51(Pt 5), 682 - 94 TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant; Fonze E; beta-Lactamases are bacterial enzymes which catalyse the hydrolysis of the beta-lactam ring of penicillins, cephalosporins and related compounds, thus inactivating these antibiotics . The crystal structure of the TEM1 beta-lactamase has been determined at 1.9 A resolution by the molecular-replacement method, using the atomic coordinates of two homologous beta-lactamase refined structures which show about 36% strict identity in their amino-acid sequences and 1.96 A r.m.s . deviation between equivalent Calpha atoms . The TEM1 enzyme crystallizes in space group P2(1)2(1)2(1) and there is one molecule per asymmetric unit . The structure was refined by simulated annealing to an R-factor of 15.6% for 15 086 reflections with I >/= 2sigma(I) in the resolution range 5.0-1.9 A . The final crystallographic structure contains 263 amino-acid residues, one sulfate anion in the catalytic cleft and 135 water molecules per asymmetric unit . The folding is very similar to that of the other known class A beta-lactamases . It consists of two domains, the first is formed by a five-stranded beta-sheet covered by three alpha-helices on one face and one alpha-helix on the other, the second domain contains mainly alpha-helices . The catalytic cleft is located at the interface between the two domains . We also report the crystallographic study of the TEM S235A mutant . This mutation of an active-site residue specifically decreases the acylation rate of cephalosporins . This TEM S235A mutant crystallizes under the same conditions as the wild-type protein and its structure was refined at 2.0 A resolution with an R value of 17.6% . The major modification is the appearance of a water molecule near the mutated residue, which is incompatible with the OG 235 present in the wild-type enzyme, and causes very small perturbations in the interaction network in the active site. J Clin Microbiol, 2004 Aug, 42(8), 3888 - 90 Mucoid nitrate-negative Moraxella nonliquefaciens from three patients with chronic lung disease; Davis JM et al.; Mucoid strains of Moraxella nonliquefaciens were recovered from the sputa of three indigenous Australians with chronic lung disease . These atypical strains failed to reduce nitrate, and one strain produced beta-lactamase . While the mucoid phenotype of M . nonliquefaciens has rarely been reported, the mucoid nitrate-negative biovar has never been previously reported. Infection, 2004 Aug, 32(4), 246 - 8 Failure of ceftriaxone in an intravenous drug user with invasive infection due to Ralstonia pickettii; Zellweger C et al.; We report a case of septic arthritis due to Ralstonia pickettii in an intravenous drug user with unfavorable clinical course under antibiotic therapy with ceftriaxone despite in vitro susceptibility to the drug . The treatment failure may have been due to a discrepancy between in vitro and in vivo susceptibility of R . pickettii, or to resistance development mediated by a recently described inducible beta-lactamase. J Bacteriol, 2004 Aug, 186(16), 5486 - 95 Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter; Charpentier X et al.; Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS) . Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton . Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains . In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase . Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM . We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells . Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner . A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation . In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation . In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells. Curr Med Chem, 2004 Jul, 11(14), 1813 - 35 Strategies for the stereocontrolled formation of oxygen analogues of penicillins and cephalosporins; Lysek R et al.; The synthesis of oxacephalotin and oxacephamandol, which are more active than natural, sulfur-containing congeners, and the isolation of clavulanic acid, a potent inhibitor of beta-lactamase enzymes, directed attention of many academic and industrial laboratories the synthesis of oxygen analogues of penicillins and cephalosporins . The present review focuses attention on the problem of stereocontrol in the formation of a desired configuration of the bridgehead carbon atom in the title compounds . Five feasible synthetic methods leading to the basic skeletons of clavams and 5-oxacephams are discussed . Three of them involve the nucleophilic substitution at C-4 of the azetidin- 2-ones performed as inter- or intramolecular process and the remaining two involve cycloaddition reactions between ketenes and iminoethers, or between vinyl ethers and isocyanates . Owing to the general application, stereospecificity and high asymmetric induction, the last method seems to be most advantageous . The weak point of the nucleophilic substitution methodology is that a nucleophile approaches the 3-substituted azetidin-2-one ring preferentially anti to the existing substituent and in the case where there is no substituent at C-3, that the stereoselectivity of formation of the new chirality center at C-4 is low . All discussed methods are illustrated by the examples taken from the literature. J Chem Phys, 2004 May 1, 120(17), 8039 - 52 Adapting the nudged elastic band method for determining minimum-energy paths of chemical reactions in enzymes; Xie L et al.; Optimization of reaction paths for enzymatic systems is a challenging problem because such systems have a very large number of degrees of freedom and many of these degrees are flexible . To meet this challenge, an efficient, robust and general approach is presented based on the well-known nudged elastic band reaction path optimization method with the following extensions: (1) soft spectator degrees of freedom are excluded from path definitions by using only inter-atomic distances corresponding to forming/breaking bonds in a reaction; (2) a general transformation of the distances is defined to treat multistep reactions without knowing the partitioning of steps in advance; (3) a multistage strategy, in which path optimizations are carried out for reference systems with gradually decreasing rigidity, is developed to maximize the opportunity of obtaining continuously changing environments along the path . We demonstrate the applicability of the approach using the acylation reaction of type A beta-lactamase as an example . The reaction mechanism investigated involves four elementary reaction steps, eight forming/breaking bonds . We obtained a continuous minimum energy path without any assumption on reaction coordinates, or on the possible sequence or the concertedness of chemical events . We expect our approach to have general applicability in the modeling of enzymatic reactions with quantum mechanical/molecular mechanical models . J Antimicrob Chemother, 2004 Aug, 54(2), 348 - 53 Epub 2004 Jul 14. Analysis of sequence variation among smeDEF multi drug efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophilia isolates; Gould VC et al.; OBJECTIVES: To determine the level of variation in the smeDEF efflux pump and smeT transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical Stenotrophomonas maltophilia isolates . METHODS: smeDEF sequencing used a PCR genome walking approach . Determination of the sequence surrounding smeDEF used a flanking primer PCR method and specific primers anchored in smeD or smeF together with random primers . RESULTS: smeDEF is chromosomal and located in the same position in the chromosome in all three subgroups of isolates . Flanking smeD is a gene, smeT, encoding a putative transcriptional repressor for smeDEF . Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic beta-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint . The smeD-smeT intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates . CONCLUSIONS: These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, S . maltophilia, based on genotypic properties . Isolate D457, in which most work concerning smeDEF expression has been performed, does not fall into S . maltophilia subgroup A, which is the most typical. J Gen Virol, 2004 Jul, 85(Pt 7), 1867 - 75 Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon; Graziani R et al.; An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture . It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B . However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins . The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons . This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture . With the use of a beta-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated . Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons . This feature was characterized as a dominant negative effect . Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon . In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons. Appl Biochem Biotechnol, 2004 May, 117(2), 115 - 22 The "Bringer" strategy: a very fast and highly efficient method for construction of mutant libraries by error-prone polymerase chain reaction of ring-closed plasmids; Bichet A et al.; Random mutagenesis constitutes a keystone in many strategies of directed evolution of biocatalysts and is often done by error-prone polymerase chain reaction (epPCR) . Traditionally, the epPCR-generated DNA fragments are then subcloned into an expression vector to obtain a mutant library, which in turn is transformed into a suited host and screened for mutants that display the desired property . However, the vast majority of epPCR-generated fragments generally are lost during the subcloning step, making it the bottleneck in the mutant library construction procedure . Here we report a rapid and convenient strategy based on the epPCR amplification of a ring-closed expression plasmid that contains the gene of interest; after a DpnI digest the product of the epPCR reaction constitutes the mutant library and can be used directly for screening procedures . Primers binding to the beta-lactamase gene were chosen to allow application of the strategy to as broad a range of target plasmids as possible . The functionality of this approach was demonstrated by mutating the alpha-peptide coding region of the lacZ gene. J Biol Chem, 2004 Aug 6, 279(32), 33630 - 8 Epub 2004 May 24. Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis; Mercuri PS et al.; The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems . FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography . In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics . The activity of H121A is dependent on zinc ion concentration . The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme . Tyr156 is not essential for binding and hydrolysis of the substrate . Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems . The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins . A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex. Infect Immun, 2004 Jun, 72(6), 3336 - 43 Recombinant Mycobacterium bovis BCG expressing the Sm14 antigen of Schistosoma mansoni protects mice from cercarial challenge; Varaldo PB et al.; The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall . The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability . The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14 . Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S . mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli . The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries. J Bacteriol, 2004 Jun, 186(11), 3431 - 8 Two oligopeptide-permease-encoding genes in the clavulanic acid cluster of Streptomyces clavuligerus are essential for production of the beta-lactamase inhibitor; Lorenzana LM et al.; orf7 (oppA1) and orf15 (oppA2) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical . These proteins show sequence similarity to periplasmic oligopeptide-binding proteins . Mutant S . clavuligerus oppA1::acc, disrupted in oppA1, lacks clavulanic acid production . Clavulanic acid production is restored by transformation with plasmid pIJ699-oppA1, which carries oppA1, but not with the multicopy plasmid pIJ699-oppA2, which carries oppA2 . The mutant S . clavuligerus oppA2::aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5) . Clavulanic acid production at low levels is restored in the oppA2-disrupted mutants by transformation with plasmid pIJ699-oppA2, but it is not complemented by the multicopy plasmid pIJ699-oppA1 . Both genes encode oligopeptide permeases with different substrate specificities . The disrupted S . clavuligerus oppA2::aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes . The null S . clavuligerus oppA1::acc and S . clavuligerus oppA2::aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptide-binding proteins. Acc Chem Res, 2004 May, 37(5), 297 - 303 Beta-sultams-mechanism of reactions and use as inhibitors of serine proteases; Page MI; beta-Sultams are reactive sulfonyl analogues of beta-lactams and show enormous rate enhancements over analogous reactions of sulfonamides . N-Acyl beta-sultams undergo S-N rather than C-N fission, although alpha-alkenyl substituents direct nucleophilic attack to the acyl center . They also inactivate serine enzymes such as elastase and beta-lactamase by sulfonylation of the active site serine . Structure-activity relationships are used to identify differences in transition state structures. J Biomol Screen, 2004 Apr, 9(3), 186 - 95 Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems; Kornienko O et al.; Cell-based beta-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 microL total assay volume in a 3456-well microplate . Studies were done to evaluate both receptor agonists and antagonists . The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC(50)/IC(50) precision . These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h . The instrumentation used and practical aspects of the assay development are discussed. J Clin Microbiol, 2004 May, 42(5), 2203 - 6 Use of beta-lactamase inhibitors in disk tests to detect plasmid-mediated AmpC beta-lactamases; Black JA et al.; Seeking a simple disk test for detection of organisms producing plasmid-mediated AmpC beta-lactamases, we evaluated the diagnostic utility of the beta-lactamase inhibitors 48-1220 (Ro 48-1220) and LN-2-128 . Using NCCLS disk methodology, inhibition zone diameters were determined for five beta-lactam antibiotics tested alone and in combination with 20 microg of either 48-1220 or LN-2-128 . Using an increase of > or =4 mm in zone diameter in the presence of an inhibitor as a positive test, cefotetan with LN-2-128 and 48-1220 was adequate for the detection of organisms producing plasmid-mediated AmpCs (specificity of 90% and sensitivity of 100%). Nucleic Acids Res, 2004 Apr 28, 32(8), 2336 - 41 Print 2004. A novel replicating circular DNAzyme; Chen F et al.; 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage . However, the dependence on exogenous delivery limits its applications . The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector . By cloning the 10-23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz7 and C-Dz482, targeting the beta-lactamase mRNA . These circular DNAzymes showed in vitro catalytic efficiencies (kcat/K(M)) of 7.82 x 10(6) and 1.36 x 10(7) M(-1) x min(-1), respectively . Their dependence on divalent metal ions is similar to that found with linear 10-23 DNAzyme . Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting beta-lactamase and bacterial growth . This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications. Folia Microbiol (Praha), 2004, 49(1), 71 - 4 Double-disk synergy test positivity in Stenotrophomonas maltophilia clinical strains; Hejnar P et al.; The double-disk synergy test (DDST) using Mueller-Hinton agar and antibiotic disks with centrally positioned disks of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam and, at a center-to-center distance of 25-30 mm, 2-4 disks with 10 various beta-lactam antibiotics per one plate was performed in 58 clinical isolates of Stenotrophomonas maltophilia to determine the effectivity of 3 beta-lactamase inhibitors . When tested with clavulanate as the central beta-lactamase inhibitor synergic action on tested strains was the most frequent with aztreonam (81.0% of strains), cefoperazone (63.8%), and cefepime (60.3%) . With sulbactam the synergic action, i.e . DDST positivity, was high in the case of cefoperazone (15.5%), ampicillin, aztreonam and piperacillin (8.6% each); with tazobactam it was the most frequent with aztreonam (53.4%), cefoperazone (44.8%) and cefepime (37.9%) . No synergy was demonstrated after application of meropenem regardless of the kind of beta-lactamase inhibitor used . In 58 strains of S . maltophilia, 55 different profiles of DDST positivity were found . The results confirm that clavulanate is the most effective inhibitor of S . maltophilia beta-lactamases . The utilization of DDST (performed in the recommended way) for the typization of strains Stenotrophomonas species and for the estimation of potential effectiveness combinations of beta-lactams with beta-lactamase inhibitors for the therapy of stenotrophomonade infections was suggested. Antimicrob Agents Chemother, 2004 May, 48(5), 1670 - 5 Genetic and biochemical characterization of a chromosome-encoded carbapenem-hydrolyzing ambler class D beta-lactamase from Shewanella algae; Heritier C et al.; A chromosome-encoded beta-lactamase gene from Shewanella algae clinical isolate KB-1 was cloned and expressed in Escherichia coli . It encoded the Ambler class D enzyme OXA-55, sharing less than 55% identity with any other oxacillinases . Although conferring a narrow-spectrum beta-lactam resistance phenotype, OXA-55 had carbapenem-hydrolyzing activity that mirrored the reduced susceptibility to imipenem observed in S . algae KB-1 . Very similar oxacillinases were found in other S . algae isolates. Antimicrob Agents Chemother, 2004 May, 48(5), 1454 - 60 Role of a mutation at position 167 of CTX-M-19 in ceftazidime hydrolysis; Kimura S et al.; CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum beta-lactamase, which differs from the majority of CTX-M-type beta-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime . To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the beta-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed . We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 micro g/ml) . Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime (k(cat)/K(m), 1.47 x 10(6) s(-1) M(-1)), cefoxitin (k(cat)/K(m), 62.2 s(-1) M(-1)), and aztreonam (k(cat)/K(m), 1.34 x 10(3) s(-1) M(-1)) are good substrates and that imipenem (k(+2)/K, 1.20 x 10(2) s(-1) M(-1)) is a poor substrate . However, CTX-M-18 and CTX-M-19 exhibited too high a K(m) value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity (k(cat)) . Comparison of the MICs with the catalytic efficiency (k(cat)/K(m)) of these enzymes showed that some beta-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation . Using the previously reported crystal structure of the Toho-1 beta-lactamase, which belongs to the CTX-M-type beta-lactamase group, we have suggested characteristic interactions between the enzymes and the beta-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling . Aminothiazole-bearing beta-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of beta-lactams. J Mol Biol, 2004 Feb 20, 336(3), 763 - 74 Effect of crowding on protein-protein association rates: fundamental differences between low and high mass crowding agents; Kozer N et al.; Physiological media constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo . Measuring protein-protein interaction in crowded solutions can mimic this environment . In this work we follow the process of protein-protein association and its rate constants (k(on)) of the beta-lactamase (TEM)-beta-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents . In all crowded solutions (0-40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k(on), but not k(off) values, were found to be slower as compared to buffer . However, there is a fundamental difference between low and high mass crowding agents . In the presence of low mass crowding agents and Haemaccel k(on) depends inversely on the solution viscosity . In high mass polymer solutions k(on) changes only slightly, even at viscosities 12-fold higher than water . The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (Lp) . Polymers that are long enough to form a flexible coil (L/Lp > 2) behave as high molecular mass polymers and those who are unable to do so (L/Lp < 2) behave as low molecular mass polymers . We concluded that although polymers solution are crowded, this property is not uniform; i.e . there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment . This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton . To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k(on) of wt-TEM and three BLIP analogs with up to 100-fold increased values of k(on) due to electrostatic steering . Faster associating BLIP variants keep their relative advantage in all crowded solutions, including Haemaccel . This result suggests that faster associating protein complexes keep their advantage also in complex environment. Assay Drug Dev Technol, 2003 Dec, 1(6), 789 - 800 A beta-lactamase-dependent Gal4-estrogen receptor beta transactivation assay for the ultra-high throughput screening of estrogen receptor beta agonists in a 3456-well format; Peekhaus NT et al.; Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta . Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis . Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes . The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology . In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol . To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta . The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay . The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists . Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta. Assay Drug Dev Technol, 2003 Dec, 1(6), 777 - 87 Miniaturization of cell-based beta-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptors; Chin J et al.; Activation of liver X receptors (LXRs) induces reverse cholesterol transport and increases high-density lipoprotein cholesterol in vivo . Here, we describe novel, functional, homogeneous cell-based fluorescence resonance energy transfer assays for identifying agonists of LXRs using beta-lactamase as the reporter gene . Stable Chinese hamster ovary cell lines expressing LXRalpha-GAL4 or LXRbeta-GAL4 fusion proteins that regulate beta-lactamase transcription from upstream 7 x UAS GAL4 DNA binding sequences were generated and characterized . Synthetic and natural ligands of LXR dose-dependently activated the expression of beta-lactamase in a subtype-specific manner . These assays were used to demonstrate that a 1-pyridyl hydantoin small molecule LXR synthetic ligand specifically activates LXRalpha receptors . The beta-lactamase assays were optimized for cell density, dimethyl sulfoxide sensitivity, and time of agonist stimulation . Clonal LXRbeta-GAL4-beta-lactamase cells were miniaturized into an ultra high throughput (3456-well nanoplates) screening format. Assay Drug Dev Technol, 2003 Dec, 1(6), 767 - 76 A one-arm homologous recombination approach for developing nuclear receptor assays in somatic cells; Qureshi SA et al.; Drug discovery is in need of technologies that enable investigators to develop cell-based assays that accurately reflect the functional consequence of small molecule intervention on biological processes . Here, we describe a strategy that uses both one-arm homologous recombination and the beta-lactamase (BLA) reporter system, a sensitive and robust transcriptional reporter for gene activation . We demonstrate that this powerful approach can be utilized for developing cell-based assays for the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in HEK293 somatic cells . Specifically, one-arm homologous recombination was used to introduce the GAL4 DNA-binding domain (GAL4DBD) in the GR and MR genomic loci such that a chimeric GAL4DBD-GR (ligand-binding domain) {GAL4DBD-GR(LBD)} and GAL4DBD-MR(LBD) transcript is produced from the strong CMV promoter in HEK293 cells previously stably transfected with the UAS(GAL4)-BLA reporter construct . Dexamethasone- and aldosterone-responding BLA-positive cells were isolated by fluorescence-activated cell sorting, and then further expanded into separate cell lines . The sensitivity and robustness of the resulting GR and MR assays are demonstrated by the fact that the addition of dexamethasone and aldosterone to the two transgenic clonal cell lines for 16 h results in high Z' values (>0.8) and EC(50) values of 1 and 0.3 nM, respectively . These assays illustrate the flexibility of this technology to generate high-performance cellular assays for nuclear receptor targets without the need for target-specific cDNA. Methods Mol Biol, 2004, 261, 411 - 26 Mapping biochemical networks with protein-fragment complementation assays; Remy I et al.; Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes . Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells . The experimental strategy is based on protein-fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction . A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, and so on) . In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and beta-lactamase. J Mol Biol, 2004 Mar 5, 336(5), 1283 - 91 Allosteric inhibition through core disruption; Horn JR et al.; Although inhibitors typically bind pre-formed sites on proteins, it is theoretically possible to inhibit by disrupting the folded structure of a protein or, in the limit, to bind preferentially to the unfolded state . Equilibria defining how such molecules act are well understood, but structural models for such binding are unknown . Two novel inhibitors of beta-lactamase were found to destabilize the enzyme at high temperatures, but at lower temperatures showed no preference for destabilized mutant enzymes versus stabilized mutants . X-ray crystal structures showed that both inhibitors bound to a cryptic site in beta-lactamase, which the inhibitors themselves created by forcing apart helixes 11 and 12 . This opened up a portion of the hydrophobic core of the protein, into which these two inhibitors bind . Although this binding site is 16 A from the center of the active site, the conformational changes were transmitted through a sequence of linked motions to a key catalytic residue, Arg244, which in the complex adopts conformations very different from those in catalytically competent enzyme conformations . These structures offer a detailed view of what has heretofore been a theoretical construct, and suggest the possibility for further design against this novel site. Biochemistry, 2004 Mar 30, 43(12), 3570 - 81 Correlation between catalytic efficiency and the transcription read-out in chemical complementation: a general assay for enzyme catalysis; Sengupta D et al.; High-throughput assays for enzyme catalysis that can be applied to a broad range of chemical reactions are key to advances in directed evolution and proteomics . Recently, we reported such a general assay, chemical complementation, which links enzyme catalysis to reporter gene transcription in vivo using the yeast three-hybrid assay . In this proof-of-principle experiment, it was shown that a wild-type beta-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen . Ideally, however, such an assay should be able to distinguish enzymes based on their catalytic activity . Thus, here, we set out to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level of transcription activation in the chemical complementation assay . First, the reaction mechanism for the cleavage of the beta-lactam substrate used in the chemical complementation proof-of-principle experiment was determined . Then a series of beta-lactamase variants was designed to span several orders of magnitude in k(cat)/K(m) . The activity of each variant was determined both in vitro using purified enzyme and in vivo in the chemical complementation transcription assay . Beta-lactamase variants spanning three-orders of magnitude in k(cat)/K(m) could be distinguished in the assay, and the catalytic efficiency of each variant correlated with its level of transcription activation in vivo . These results establish the suitability of chemical complementation for the directed evolution of enzymes with improvements in catalytic activity and for profiling the relative substrate specificities of a group of enzymes in proteomics applications. Oligonucleotides, 2003, 13(6), 427 - 33 The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli; Dryselius R et al.; Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity . Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria . However, many design uncertainties remain, including the difficult question of target sequence selection . In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase . The results from this scan pointed to the start codon region as most sensitive to inhibition . To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene . For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective . These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism . Therefore, although other design parameters are also important, the start codon region in E . coli mRNA is the most reliable target site for antisense PNAs. Clin Microbiol Infect, 2004 Mar, 10(3), 234 - 41 Mechanisms of reduced susceptibility to amoxycillin-clavulanic acid in Escherichia coli strains from the health region of Tortosa (Catalonia, Spain); Perez-Moreno MO et al.; This study investigated the mechanisms involved in reduced susceptibility to amoxycillin-clavulanic acid and the prevalence of enzymes compatible with inhibitor-resistant TEM (IRT) beta-lactamases produced by Escherichia coli isolates from patients in north-eastern Spain . The resistance mechanisms of 158 strains showing resistance or intermediate resistance to amoxycillin-clavulanic acid among 1122 ampicillin-resistant clinical isolates of E . coli were assessed on the basis of their beta-lactam resistance phenotypes . beta-Lactamases produced by strains showing resistant phenotypes suggestive of inhibitor-resistant penicillinase production were characterised by their isoelectric point . Specific activity and the concentration of clavulanic acid required to inhibit beta-lactamase activity by 50% (IC50) were determined in strains harbouring enzymes that focused at pI 5.2 or 5.4 in order to achieve presumptive identification of IRT beta-lactamases . Resistance phenotypes were consistent with overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases in 56 strains, with AmpC cephalosporinase hyperproduction in 46 strains, and with production of inhibitor-resistant penicillinases in 49 strains . Of the latter isolates, 17 produced moderately high or high levels of enzymes co-focusing with TEM-1, 17 produced enzymes co-focusing with OXA-1 (n = 12) or with PSE-1 (n = 5), either alone or in association with TEM-1, while only 15 produced enzymes with a phenotype characteristic of IRT beta-lactamases . It was concluded that resistance to amoxycillin-clavulanic acid in E . coli isolates from this area was mainly associated with presumptive overproduction of TEM-1, TEM-2 or SHV-1 beta-lactamases (46%) or of AmpC cephalosporinase (29%), while the occurrence of enzymes categorised as IRT beta-lactamases was unusual (9.5%). J Infect Chemother, 2004 Feb, 10(1), 62 - 4 The combination of aztreonam and cefozopran against Stenotrophomonas maltophilia; Kataoka D et al.; Aztreonam is suited for combination chemotherapy because it could be a potent Beta-lactamase inhibitor . We designed a study to show the inhibitory activity of aztreonam, using Stenotrophomonas maltophilia, which produces both carbapenemase L1 and penicillinase L2 . Aztreonam showed considerable synergy with cefpirome and contributed to a decrease in minimum inhibitory concentrations of cefozopran . In further examinations, the mean viable bacterial counts in cultures treated with aztreonam-cefozopran were 1 log lower than those in cultures treated with cefozopran alone . These results confirm that inhibition of penicillinase L2 occurred . We hope that a combination chemotherapy using aztreonam and cefozopran will be used to prevent the emergence of penicillinase-producers. Protein Eng, 2003 Dec, 16(12), 1025 - 34 Using a residue clash map to functionally characterize protein recombination hybrids; Saraf MC et al.; In this article, we introduce a rapid, protein sequence database-driven approach to characterize all contacting residue pairs present in protein hybrids for inconsistency with protein family structural features . This approach is based on examining contacting residue pairs with different parental origins for different types of potentially unfavorable interactions (i.e . electrostatic repulsion, steric hindrance, cavity formation and hydrogen bond disruption) . The identified clashing residue pairs between members of a protein family are then contrasted against functionally characterized hybrid libraries . Comparisons for five different protein recombination studies available in the literature: (i) glycinamide ribonucleotide transformylase (GART) from Escherichia coli (purN) and human (hGART), (ii) human Mu class glutathione S-transferase (GST) M1-1 and M2-2, (iii) beta-lactamase TEM-1 and PSE-4, (iv) catechol-2,3-oxygenase xylE and nahH, and (v) dioxygenases (toluene dioxygenase, tetrachlorobenzene dioxygenase and biphenyl dioxygenase) reveal that the patterns of identified clashing residue pairs are remarkably consistent with experimentally found patterns of functional crossover profiles . Specifically, we show that the proposed residue clash maps are on average 5.0 times more effective than randomly generated clashes and 1.6 times more effective than residue contact maps at explaining the observed crossover distributions among functional members of hybrid libraries . This suggests that residue clash maps can provide quantitative guidelines for the placement of crossovers in the design of protein recombination experiments. Antimicrob Agents Chemother, 2004 Mar, 48(3), 1032 - 3 In vitro evolution predicts that the IMP-1 metallo-beta-lactamase does not have the potential to evolve increased activity against imipenem; Hall BG; In vitro evolution was used to predict whether the IMP-1 metallo-beta-lactamase has the potential to evolve an increased ability to confer resistance to imipenem . Screening of eight libraries containing 9.8 x 10(6) +/- 1.4 x 10(6) (mean +/- standard error) variants per library, with an average of 1.2 mutations per variant, detected no increased resistance to imipenem . The results predict, with >99.9% confidence, that even under intense selection the IMP-1 beta-lactamase will not evolve to confer increased resistance to imipenem. Methods Mol Biol, 2004, 263, 333 - 44 Fluorescence resonance energy transfer-based HIV-1 virion fusion assay; Cavrois M et al.; The fluorescence resonance energy transfer (FRET)-based HIV-1 virion fusion assay exploits the incorporation of beta-lactamase-Vpr chimeric proteins into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a marker of fusion . This transfer can be monitored by the enzymatic cleavage of the CCF2-AM dye, a fluorescent substrate of beta-lactamase (BlaM), loaded into the target cells . BlaM cleavage of the beta-lactam ring in CCF2-AM prevents the FRET between the coumarin and fluorescein moieties of the dye . This cleavage changes the fluorescence emission spectrum of CCF2-AM from green (520 nm) to blue (447 nm), and thus permits detection of fusion by fluorescence microscopy, flow cytometry, or UV photometry . This assay is simple and rapid to perform, and exhibits high sensitivity and specificity . Importantly, it can be applied to study HIV-1 virion fusion in primary cells and can be combined with immunostaining for subset discrimination in heterogeneous target cell populations . Finally, the assay can also be adapted to study fusion mediated by the envelope proteins from other viruses through the construction of HIV-1 pseudotypes. Biochemistry, 2004 Feb 17, 43(6), 1715 - 23 Folding of an abridged beta-lactamase; Santos J et al.; The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B . licheniformis exo small beta-lactamase (ES-betaL) have been measured . ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no enzymic activity . Deletion of the last 14 residues produces ES-betaL(C)(Delta)(14), which is 0.1% active . The enzyme lacking nine residues (ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme . Although ES-betaL(C)(Delta)(9) folds properly, it does so 4 orders of magnitude slower than ES-betaL, making possible the isolation and characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)) . Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow folding . Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence . The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level . The results are compared with other examples of this kind in the folding literature. J Biol Chem, 2004 May 7, 279(19), 19494 - 501 Epub 2004 Feb 02. Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition; Pagan-Rodriguez D et al.; The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections . Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds . In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor . Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry . The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase . These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition . Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation . By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme . After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions. J Mol Biol, 2004 Feb 6, 336(1), 263 - 73 Creation of an allosteric enzyme by domain insertion; Guntas G et al.; Two allosteric enzymes have been created by the covalent linkage of non-interacting, monomeric proteins with the prerequisite effector-binding and catalytic functionalities, respectively . This was achieved through a combinatorial process called random domain insertion . The fragment of the TEM-1 beta-lactamase gene coding for the mature protein lacking its signal sequence was randomly inserted into the Escherichia coli maltose-binding protein (MBP) gene to create a domain insertion library . This library's diversity derived both from the site of insertion and from a distribution of tandem duplications or deletions of a portion of the MBP gene at the insertion site . From a library of approximately 2 x 10(4) in-frame fusions, approximately 800 library members conferred a phenotype to E.coli cells that was consistent with the presence of bifunctional fusions that could hydrolyze ampicillin and transport maltose in E.coli . Partial screening of this bifunctional sublibrary resulted in the identification of two enzymes in which the presence of maltose modulated the rate of nitrocefin hydrolysis . For one of these enzymes, the presence of maltose increased k(cat) by 70% and k(cat)/K(m) by 80% and resulted in kinetic parameters that were almost identical to TEM-1 beta-lactamase . Such an increase in activity was only observed with maltooligosaccharides whose binding to MBP is known to induce a conformational change . Modulation of the rate of nitrocefin hydrolysis could be detected at maltose concentrations less than 1 microM . Intrinsic protein fluorescence studies were consistent with a conformational change being responsible for the modulation of activity. Vaccine, 2004 Jan 26, 22(5-6), 740 - 6 Adjuvant activity of Mycobacterium bovis BCG expressing CRM197 on the immune response induced by BCG expressing tetanus toxin fragment C; Mazzantini RP et al.; In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M . fortuitum beta-lactamase promoter, pBlaF* . Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol . Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile . Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response . Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin . Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin . Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases. Appl Environ Microbiol, 2004 Jan, 70(1), 494 - 7 Improved method for polynucleotide probe-based cell sorting, using DNA-coated microplates; Zwirglmaier K et al.; We developed an improved method for cultivation-independent sorting of bacterial cells . The technique is based on labeling the target cells by in situ hybridization with polynucleotide transcript probes . Due to the probes' length, part of the probe remains outside the cell and can subsequently be used to capture the cells . Target cells are immobilized during a second hybridization step in microplates that are coated with DNA that is complementary to the probe sequence . The method was applied successfully to artificial mixtures of cells with polynucleotide probes targeting either rRNA, a plasmid-borne beta-lactamase gene, or a chromosome-borne glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene . Cells could be separated based on phylogenetic parameters (using rRNA-targeted probes) as well as on other DNA-encoded traits. Chem Commun (Camb), 2003 Dec 21, (24), 2998 - 9 Colloidal stable silica encapsulated nano-magnetic composite as a novel bio-catalyst carrier; Gao X et al.; A colloidal stable silica-encapsulated magnetic nano-composite of a controlled dimension is, for the first time, employed to carry beta-lactamase via chemical linkage on the silica overlayer: activity study reflects that this new type of immobilisation allows site (enzyme) isolation, accessibility as good as free enzyme and recovery & reusability upon application of magnetic separation. Yi Chuan Xue Bao, 2003 Aug, 30(8), 730 - 6 {Construction and verification of the effectiveness of pMBL: a cloning vector of exported proteins encoding genes}; Yu XP et al.; The beta-lactamase was used as the reporter of expression and transmembrane secretion in this paper . A fragment of Amp resistance gene encoding the mature part of beta-lactamase (delta P delta SP Amp, i.e . without promoter and signal peptide coding sequences) was amplified from pUC18 vector . The upstream primer has BglII, BclI, BamHI in three reading frame respectively, in order to in frame fuse and express target genes together with the downstream reporter in finally constructed vector . Meanwhile, pET-28 was digested with the restriction enzymes BglII and Bst1107 I . The 2.8 kb fragment with replication origin, Kan resistance gene and MCS was recovered, filled, self-ligated and resulted in a plasmid pKan-B . The Bgl II site on pKan-B was then filled and the plasmid pKan was obtained . The delta P delta SP Amp gene, which was first cloned into pGEM-T-EASY vector, was inserted into pKan between EcoR I and XbaI sites . A plasmid pMBL-E was selected, with which the bacteria host could grew on Kan plate but not on plate with both Amp and Kan . An EcoRI site beside HindIII on the plasmid pMBL-E was then filled, and the plasmid pMBL, a cloning vector of the exported proteins encoding genes was finally obtained . Both results of the restriction enzyme digestion and sequencing demonstrated the correctness of the construction . The Tet resistance gene, a transmemebrane protein encoding gene, was applied to verify the effectiveness of the reporter in the vector . Cut with EcoRI and BamHI, a 375 bp fragment including promoter and 96 animo acids coding sequence (including signal peptide) of Tet was obtained from pBR322 vector . The fragment was then ligated to the vector pMBL which had been cut with both enzymes of EcoRI and BglI, or EcoRI and BclI, or EcoRI and BamHI (as 0, +1, +2 respectively of the beta-lactamase gene reading frame) . Kan and Amp double resistant colonies only grew with the EcoRI and BglII combination (0 position) . Restriction enzyme digestion and sequencing results of the recombinant plasmid showed that Tet resistance gene, which promoted the expression and transmembrane secretion of downstream beta-lactamase, was inserted in a correct reading frame into the vector . Thus, the results verified the effectiveness of the constructed vector pMBL, which may be used effectively to clone genes encoding exported proteins with promoters and signal peptide sequences. Curr Opin Struct Biol, 2003 Dec, 13(6), 709 - 15 Sub-Angstrom resolution enzyme X-ray structures: is seeing believing? Vrielink A, Sampson N. Recent technical advances in crystallographic analysis, particularly highly focused and high brilliance synchrotron beam lines, have significantly improved the resolutions that are attainable for many macromolecular crystal structures . The Protein Data Bank contains an increasing number of atomic resolution structures, which are providing a wealth of structural information that was not previously visible in lower resolution electron density maps . Here, we review the importance of visualizing hydrogen atoms and multiple sidechain conformations or anisotropy, as well as substrate strain, at sub-Angstrom resolution . The additional structural features that are visible in the electron density maps as a result of atomic resolution data provide a better understanding of the catalytic mechanisms of cholesterol oxidase, ribonuclease A, beta-lactamase, serine proteases, triosephosphate isomerase and endoglucanase. Biochemistry, 2003 Dec 16, 42(49), 14483 - 91 Thermodynamic cycle analysis and inhibitor design against beta-lactamase; Roth TA et al.; Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and cephalosporins . Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes . To understand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamic cycle experiments were undertaken . An unexpected hydrogen bond between the nonconserved Asn289 and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nM inhibitor (compound 1) in complex with AmpC beta-lactamase . To investigate the energy of this hydrogen bond, the mutant enzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2) . The differential affinity of the four different protein and analogue complexes indicates that the carboxylate-amide hydrogen bond contributes 1.7 kcal/mol to overall binding affinity . Synthesis of an analogue of 1 where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bond energy, consistent with the importance of the ionic nature of this hydrogen bond . To investigate the structural bases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 A, respectively . These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds . The mutant enzymes L119A and L293A were made to investigate the interaction between a phenyl ring in 1 and these residues . Whereas deletion of the phenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does not come through interaction with the leucines, despite the close contact in the structure . The energies of these interactions provide key information for the design of improved inhibitors against beta-lactamases . The high magnitude of the ion-dipole interaction between Asn289 and the carboxylate of 1 is consistent with the idea that ionic interactions can provide significant net affinity in inhibitor complexes. J Biol Chem, 2004 Feb 20, 279(8), 6650 - 7 Epub 2003 Dec 05. Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter; Just H et al.; Na+/Cl--dependent neurotransmitter transporters form constitutive oligomers . The topological arrangement is not known, but a leucine heptad repeat in transmembrane domain (TM) 2 and a glycophorin-like motif in TM6 have been proposed to stabilize the oligomer . To determine the topology, we generated versions of the human serotonin transporter (hSERT) that carried cyan or yellow fluorescent proteins at their amino and/or carboxyl terminus . Appropriate pairs were coexpressed to measure fluorescence resonance energy transfer (FRET) . Donor photobleaching FRET microscopy was employed to deduce the following arrangement: within the monomer, the amino and carboxyl termini are in close vicinity . In addition, in the oligomer, the carboxyl termini are closer to each other than the amino termini . Hence, a separate interaction domain (i.e . distinct from TM2 and TM6) must reside in the carboxyl-terminal half of hSERT . This was confirmed by expressing the amino- and carboxyl-terminal halves of hSERT . These were retained intracellularly; they also retained the coexpressed full-length transporter by forming export-deficient oligomers and, when cotransfected in all possible combinations, supported FRET . Hence, both the carboxyl and amino termini contain elements that drive oligomerization . By employing fragments comprising two neighboring TM helices, we unequivocally identified TM11/12 as a new contact site by donor photobleaching FRET and beta-lactamase protein fragment complementation assay . TM1/2 was also found to self-associate . Thus, oligomerization of hSERT involves at least two discontinuous interfaces . The currently identified interaction sites drive homophilic interactions . This is consistent with assembly of SERT oligomers in an array-like structure containing multimers of dimers. FEMS Microbiol Lett, 2003 Nov 21, 228(2), 187 - 91 Comparison of two RT-PCR methods for quantifying ampC specific transcripts in Escherichia coli strains; Corvec S et al.; In Escherichia coli, beta-lactam resistance usually depends on beta-lactamase production . AmpC chromosomal cephalosporinase hyperproduction is generally due to mutations in the ampC gene promoter . In order to study ampC expression in E . coli clinical strains, we have compared two methods: conventional and real-time reverse transcription-polymerase chain reaction (RT-PCR) . With both methods, ampC mRNA was found to be greatly increased in strains presenting -42 or -32 mutations in the ampC promoter, and moderately increased when a -11 mutation was present in the Pribnow box . Real-time RT-PCR represents a powerful tool combining amplification, fluorescent detection and analysis. J Biol Chem, 2004 Feb 13, 279(7), 5298 - 305 Epub 2003 Nov 21. Molecular characterization of a phospholipase D generating anandamide and its congeners; Okamoto Y et al.; Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors . Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities . These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues . We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human . The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the beta-lactamase fold . As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates . In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine . Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis . These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class. J Biol Chem, 2004 Feb 13, 279(7), 5685 - 92 Epub 2003 Nov 17. Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway; Caines ME et al.; The initial step in the biosynthesis of the clinically important beta-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N2-(2-carboxyethyl)arginine, a beta-amino acid . This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase . Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-A resolution . The structure was solved in two space groups, P2(1)2(1)2(1) and P2(1)2(1)2 . In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies . Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a "V" conformation as in related enzymes . A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate . The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2 . The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative beta-amino acids. Biochemistry, 2003 Nov 25, 42(46), 13386 - 92 Following the reactions of mechanism-based inhibitors with beta-lactamase by Raman crystallography; Helfand MS et al.; The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope . The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species . A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group . By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation . The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively . The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange . This feature was used to follow the kinetics of enamine buildup and decay in the crystal . Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species . The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band . Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design. Biochemistry, 2003 Nov 18, 42(45), 13152 - 9 Inhibition of class A and class C beta-lactamases by penems: crystallographic structures of a novel 1,4-thiazepine intermediate; Nukaga M et al.; A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo{2,1-c}{1,4}oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases . The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM) . High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo{2,1-c}{1,4}oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo{3.2.0}hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively . The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively . Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate . After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety . The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine . The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules . In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent . In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis . Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates. J Clin Microbiol, 2003 Nov, 41(11), 5310 - 2 Presumed endocarditis caused by BRO beta-lactamase-producing Moraxella lacunata in an infant with Fallot's tetrad; Nagano N et al.; A case of presumed endocarditis caused by Moraxella lacunata in a 15-month-old male infant with Fallot's tetrad is described . This infection may have occurred as the result of transmission of this organism between the father and his son . This is the first report of BRO beta-lactamase-producing M . lacunata causing presumed endocarditis. J Biol Chem, 2004 Jan 9, 279(2), 920 - 7 Epub 2003 Oct 22. Metal binding Asp-120 in metallo-beta-lactamase L1 from Stenotrophomonas maltophilia plays a crucial role in catalysis; Garrity JD et al.; Metallo-beta-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis . The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a beta-lactamase fold . In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics . The D120C, D120N, and D120S mutants were shown to bind 1.6 +/- 0.2, 1.8 +/- 0.2, and 1.1 +/- 0.2 mol of Zn(II) per monomer, respectively . The mutants exhibited 10- to 1000-fold drops in kcat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested . Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes . These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme . This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction. J Biol Chem, 2003 Dec 26, 278(52), 52724 - 9 Epub 2003 Oct 08. Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130; Helfand MS et al.; Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics . In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam . By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors . Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli . Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a red