J Biol Chem, 2000 Jun 30, 275(26), 19933 - 41 Syntenin-syndecan binding requires syndecan-synteny and the co-operation of both PDZ domains of syntenin; Grootjans JJ et al.; Syntenin is an adaptor-like molecule that binds to the cytoplasmic domains of all four vertebrate syndecans . Syntenin-syndecan binding involves the C-terminal part of syntenin that contains a tandem of PDZ domains . Here we provide evidence that each PDZ domain of syntenin can interact with a syndecan . Isolated or combined mutations of the carboxylate binding lysines in the inter-betaAbetaB loops and of the alphaB1 residues in either one or both the PDZ domains of syntenin all reduce syntenin-syndecan binding in yeast two-hybrid, blot-overlay, and surface plasmon resonance assays . PDZ2 mutations have more pronounced effects on binding than PDZ1 mutations, but complete abrogation of syntenin-syndecan binding requires the combination of both the lysine and the alphaB1 mutations in both the PDZ domains of syntenin . Isothermal calorimetric titration of syntenin with syndecan peptide reveals the presence of two binding sites in syntenin . Yet, unlike a tandem of two PDZ2 domains and a reconstituted PDZ1+PDZ2 tandem, a tandem of two PDZ1 domains and isolated PDZ1 or PDZ2 domains do not interact with syndecan bait . We conclude to a co-operative binding mode whereby neither of these two PDZ domains is sufficient by itself but where PDZ2 functions as a "major" or "high affinity" syndecan binding domain, and PDZ1 functions as an "accessory" or "low affinity" syndecan binding domain . The paired, but not the isolated PDZ domains of syntenin bind also strongly to the immobilized cytoplasmic domains of neurexin and B-class ephrins . By inference, these data suggest a model whereby recruitment of syntenin to membrane surfaces requires two compatible types of bait that are in "synteny" (occurring together in location) and engages both PDZ domains of syntenin . The synteny of compatible bait may result from the assemblies and co-assemblies of syndecans and other similarly suited partners in larger supramolecular complexes . In general, an intramolecular combination of PDZ domains that are weak, taken individually, would appear to be designed to detect rather than drive the formation of specific molecular assemblies. J Biol Chem, 2000 Jul 14, 275(28), 21678 - 87 Maize cap1 encodes a novel SERCA-type calcium-ATPase with a calmodulin-binding domain; Subbaiah CC et al.; A cDNA (CAP1) isolated from maize roots shares sequence identity with genes encoding P-type Ca(2+)-ATPases and restores the growth phenotype of yeast mutants defective in Ca(2+)-pumps . CAP1 was transcribed and translated in the yeast mutant . Furthermore, the membrane-integrated product formed a Ca(2+)-dependent phosphorylated intermediate and supported Ca(2+) transport . Although CAP1 shares greater sequence identity with mammalian "endoplasmic reticulum-type" Ca(2+)-pumps, it differs from these genes by having features of calmodulin (CaM)-regulated Ca(2+)-pumps . CAP1 from yeast microsomes bound CaM, and the CAP1-dependent Ca(2+) transport in yeast was stimulated by CaM . Peptides from the C terminus of CAP1 bound CaM . Anti-CAP1 antibodies specifically recognized a maize microsomal polypeptide that also bound CaM . A similar polypeptide also formed a Ca(2+)-dependent phosphoenzyme . Our results suggest that cap1 encodes a novel form of CaM-regulated Ca(2+)-ATPase in maize . CAP1 appears to be encoded by one or two genes in maize . CAP1 RNA is induced only during early anoxia, indicating that the Ca(2+)-pump may play an important role in O(2)-deprived maize cells. J Exp Med, 2000 Apr 17, 191(8), 1365 - 80 Evidence for class-specific factors in immunoglobulin isotype switching; Shanmugam A et al.; Immunoglobulin class switch recombination (SR) occurs by a B cell-specific, intrachromosomal deletional process between switch regions . We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities . The plasmids are novel in that they lack a eukaryotic origin of DNA replication . The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells . The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes . These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity . Finally, we provide evidence for two distinct switching activities which independently mediate mu-->alpha and mu-->gamma3 SR. Cancer Gene Ther, 2000 Feb, 7(2), 177 - 86 Enhanced antitumoral effect of adenovirus-mediated cytosine deaminase gene therapy by induction of antigen-presenting cells through stem cell factor/granulocyte-macrophage colony-stimulating factor gene transfer; Cao X et al.; Suicide gene therapy has been studied intensively for the treatment of cancer . A limited antitumoral effect was obtained by intratumoral injection of adenovirus harboring Escherichia coli cytosine deaminase gene (AdCD) in tumor-bearing mice followed by continuous administration of 5-fluorocytosine (5FC) . To address the drawbacks of the limited potential for the induction of antitumoral immunity by CD suicide gene therapy, we hypothesized that antigen-presenting cells (APCs) might contribute to the efficient induction of an antitumoral immune response in tumor-bearing mice undergoing suicide gene therapy . We preinjected the mice with murine stem cell factor (SCF)-encoding adenovirus (AdSCF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-encoding adenovirus (AdGM-CSF); after 7 days, the mice were inoculated with CT26 colon adenocarcinoma . AdCD was injected intratumorally into tumor-bearing mice followed by 5FC administration . The results showed that AdSCF/AdGM-CSF treatment could increase the number, surface molecule expression, and function of APCs efficiently . A more significant growth inhibition of established tumors and a prolongation of the survival period were observed in tumor-bearing mice after AdSCF/AdGM-CSF pretreatment in combination with AdCD/5FC therapy when compared with mice treated with AdSCF or AdGM-CSF in combination with AdCD/5FC, or AdCD/5FC alone (P < .01) . Cytotoxic T-lymphocyte activity was induced efficiently after the combined therapy, and mRNA of tumor necrosis factor-alpha, interleukin-4, interferon-gamma, and interleukin-2 was present in the tumor mass after combined therapy, suggesting that a more potent antitumoral response was induced by enhanced APCs . Our results demonstrated that AdSCF/AdGM-CSF pretreatment could activate APCs, and that these APCs could present the tumor antigens released from AdCD/5FC-killed tumor cells and activate the antitumoral response of the host, thus increasing the therapeutic efficiency of suicide gene therapy. Biotechniques, 2000 Apr, 28(4), 789 - 93 Multiple epitope tagging of expressed proteins for enhanced detection; Hernan R et al.; Three FLAG epitopes have been incorporated into the mammalian expression vector pCMV-5 to create a transient expression vector, p3XFLAG-CMV-7 . The vector was designed to express FLAG fusion proteins that can be detected at tenfold lower expression levels than the current FLAG fusion protein expression system . The usefulness of this expression and detection system was demonstrated by expression of bacterial alkaline phosphatase in COS-7 cells . In addition, 3XFLAG bacterial alkaline phosphatase was expressed in Escherichia coli, purified on anti-FLAG M2 affinity gel, and detection of 500 pg of purified protein by Western blot analysis is demonstrated. Biotechniques, 2000 Apr, 28(4), 784 - 8 New positive selection system based on the parD (kis/kid) system of the R1 plasmid; Gabant P et al.; The use of vectors that are designed to allow positive selection of recombinants facilitates cloning experiments in E . coli . Using kid, a lethal gene of the R1 plasmid parD locus, we generated pKID vectors leading to high selective efficiency of recombinants (greater than 90%) . The E . coli bacterial host used to propagate these vectors produces the Kis protein, the natural antagonist of Kid . This new positive-selection system exhibits the same efficiency as the original ccdB-based selection vectors, pKIL (4) . We also show that the ccdB and kid systems are independent . This property increases the potential of plasmidic poison-antidote systems for genetic applications and opens the door to a generation of new vectors containing the two selection systems. Biotechniques, 2000 Apr, 28(4), 660 - 2, 664, 666 passim Efficient DNA subcloning through selective restriction endonuclease digestion; Spear MA; Described here is a selective restriction endonuclease digestion method that eliminates the electrophoresis step that is usually used during the subcloning of new DNA sequences into typical E . coli-based plasmids . The method increases yield while decreasing laboratory resource and time utilization . By using donor and acceptor sequences that contain unique restriction sites found only outside of the intended recombination sequences, the initial digestion products can be directly combined without electrophoresis if the ligation step is followed by a selective digestion using the unique restriction enzymes before transformation . This system is based on the several order of magnitude decrease in transformation efficiency of linearized compared to circular plasmids . As an example, this method was used to obtain recombinants between a 3.6 kb acceptor plasmid and 3.0 kb insert following one ligation reaction after the failure of nine standard reactions using similar amounts of input DNA . It is particularly applicable to situations in which low subcloning efficiencies are expected . The technique can be extended to a large percentage of planned recombinations by using nonidentical compatible cohesive or blunt-ended fragments, or site-directed mutagenesis. Biochem J, 2000 May 1, 347 Pt 3, 881 - 6 Heteroduplex DNA and ATP induced conformational changes of a MutS mismatch repair protein from Thermus aquaticus; Biswas I et al.; ATP hydrolysis by MutS homologues is required for the function of these proteins in mismatch repair . However, the function of ATP hydrolysis in the repair reaction is not very clear . We have examined the role of ATP hydrolysis in oligomerization of Thermus aquaticus (Taq) MutS protein in solution . Analytical gel filtration and cross-linking of MutS protein with disuccinimidyl suburate suggest that TaqMutS is a dimer in the presence of ATP . ATP binding and hydrolysis by TaqMutS reduces the heteroduplex-DNA binding by the protein . Using limited proteolysis we detected extensive conformational changes of the TaqMutS protein in the presence of ATP and heteroduplex DNA . Heteroduplex-DNA binding is necessary for the observed conformational changes since F39A mutant protein defective in DNA binding does not display ATP-induced conformational changes . The implications of the observed conformational changes in the MutS protein are discussed with respect to two different models proposed for the role of ATP hydrolysis by MutS in DNA mismatch repair. Biochem J, 2000 May 1, 347 Pt 3, 797 - 805 Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site; Kuo PH et al.; Subunit a of the ATP synthase F(o) sector contains a transmembrane helix that interacts with subunit c and is critical for H(+) transport activity . From a cysteine scan in the region around the essential subunit a residue, Arg-210, we found that the replacement of aGly-213 greatly attenuated ATP hydrolysis, ATP-dependent proton pumping and Delta mu(H)+-dependent ATP synthesis . Various amino acid substitutions caused similar effects, suggesting that functional perturbations were caused by altering the environment or conformation of aArg-210 . aG213N, which was particularly severe in effect, was suppressed by two second-site mutations, aL251V and cD61E . These mutations restored efficient coupling; the latter also increased ATP-dependent proton transport rates . These results were consistent with the proposed functional interaction between aArg-210 and cAsp-61, the likely carrier of the transported proton . From Arrhenius analysis of steady-state ATP hydrolytic activity, the transport mutants had large increases in the transition-state enthalpic and entropic parameters . Linear isokinetic relationships demonstrate that the transport mechanism is coupled to the rate-limiting catalytic transition-state step, which we have previously shown to involve the rotation of the gamma subunit in multi-site, co-operative catalysis. Biochem J, 2000 May 1, 347 Pt 3, 601 - 12 Role of lipids in the translocation of proteins across membranes; Van Voorst F et al.; The architecture of cells, with various membrane-bound compartments and with the protein synthesizing machinery confined to one location, dictates that many proteins have to be transported through one or more membranes during their biogenesis . A lot of progress has been made on the identification of protein translocation machineries and their sorting signals in various organelles and organisms . Biochemical characterization has revealed the functions of several individual protein components . Interestingly, lipid components were also found to be essential for the correct functioning of these translocases . This led to the idea that there is a very intimate relationship between the lipid and protein components that enables them to fulfil their intriguing task of transporting large biopolymers through a lipid bilayer without leaking their contents . In this review we focus on the Sec translocases in the endoplasmic reticulum and the bacterial inner membrane . We also highlight the interactions of lipids and proteins during the process of translocation and integrate this into a model that enables us to understand the role of membrane lipid composition in translocase function. Biochemistry, 2000 Apr 25, 39(16), 4915 - 23 The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin; Yang X et al.; Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity . Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E . coli bacterioferritin . Consistent with previous UV-visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined . The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe(2+) ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe(2+) + P(Z) --> {Fe(2)-P}(Z) + 4H(+), where P(Z) is the apoprotein of net charge Z and {Fe(2)-P}(Z) represents a diferrous ferroxidase complex . The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: {Fe(2)-P}(Z) + (1)/(2)O(2) --> {Fe(2)O-P}(Z) where {Fe(2)O-P}(Z) represents an oxidized diferric ferroxidase complex, probably a mu-oxo-bridged species as suggested by UV-visible and EPR spectrometric titration data . The third phase corresponds to mineral core formation according to the net reaction: 4Fe(2+) + O(2) + 6H(2)O --> 4FeO(OH)((core)) + 8H(+) . Iron oxidation is inhibited by the presence of Zn(2+) ions . The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn(2+)per 24mer, i.e., 2 Zn(2+) per ferroxidase center. Biochemistry, 2000 Apr 25, 39(16), 4846 - 54 Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport; Dumas F et al.; The structural and functional consequences of a mismatch between the hydrophobic thickness d(P) of a transmembrane protein and that d(L) of the supporting lipid bilayer were investigated using melibiose permease (MelB) from Escherichia coli reconstituted in a set of bis saturated and monounsaturated phosphatidylcholine species differing in acyl-chain length . Influence of MelB on the midpoint gel-to-liquid-phase transition temperature, T(m), of the saturated lipids was investigated through fluorescence polarization experiments, with 1,6-diphenyl-1,3,5-hexatriene as the probe, for varying protein/lipid molar ratio . Diagrams in temperature versus MelB concentration showed positive or negative shifts in T(m) with the short-chain lipids DiC12:0-PC and DiC14:0-PC or the long-chain lipids DiC16:0-PC and DiC18:0-PC, respectively . Theoretical analysis of the data yielded a d(L) value of 3.0 +/- 0.1 nm for the protein, similar to the 3.02 nm estimated from hydropathy profiles . Influence of the acyl chain length on the carrier activity of MelB was investigated in the liquid phase, using the monounsaturated PCs . Binding of the sugar to the transporter showed no dependence on the acyl chain length . In contrast, counterflow and Deltapsi-driven experiments revealed strong dependence of melibiose transport on the lipid acyl chain length . Similar bell-shaped transport versus acyl chain length profiles were obtained, optimal activity being supported by diC16:1-PC . On account of a d(P) value of 2.65 nm for the lipid and of various local constraints which would all tend to elongate the acyl chains in contact with the protein, one can conclude that maximal activity was obtained when the hydrophobic thickness of the bilayer matched that of the protein. Biochemistry, 2000 Apr 25, 39(16), 4831 - 7 Spin labeling analysis of structure and dynamics of the Na(+)/proline transporter of Escherichia coli; Wegener C et al.; With respect to the functional importance attributed to the N-terminal part of the Na(+)/proline transporter of Escherichia coli (PutP), we report here on the structural arrangement and functional dynamics of transmembrane domains (TMs) II and III and the adjoining loop regions . Information on membrane topography was obtained by analyzing the residual mobility of site-specifically-attached nitroxide spin label and by determination of collision frequencies of the nitroxide with oxygen and a polar metal ion complex using electron paramagnetic resonance (EPR) spectroscopy . The studies suggest that amino acids Phe45, Ser50, Ser54, Trp59, and Met62 are part of TM II while Gly39 and Arg40 are located at a membrane-water interface probably forming the cytoplasmic cap of the TM . Also Ala67 and Glu75 are at a membrane-water interface, suggesting a location close to the periplasmic ends of TMs II and III, respectively . Ser71 between these residues is clearly in a water-exposed loop (periplasmic loop 3) . Spin labels attached to positions 80, 86, and 91 show EPR properties typical for a TM location (TM III) . Leu97 may be part of a structured loop region while Ala107 is clearly located in a water-exposed loop (cytoplasmic loop 4) . Finally, spin labels attached to the positions of Asp33 and Leu37 are clearly on the surface of the transporter and are directed into an apolar environment . These findings strongly support the recently proposed 13-helix model of PutP {Jung, H., Rubenhagen, R., Tebbe, S., Leifker, K., Tholema, N., Quick, M., and Schmid, R . (1998) J . Biol . Chem . 273, 26400-26407} and suggest that TMs II and III of the transporter are formed by amino acids Ser41 to Gly66 and Ser76 to Gly95, respectively . In addition to the topology analysis, it is shown that binding of Na(+) and/or proline to the transporter alters the mobility of the nitroxide group at the positions of Leu37 and Phe45 . From these findings, it is concluded that binding of the ligands induces conformational alterations of PutP that involve at least parts of TM II and the preceding cytoplasmic loop. Biochemistry, 2000 Apr 25, 39(16), 4821 - 30 Role of metal ions in the reaction catalyzed by L-ribulose-5-phosphate 4-epimerase; Lee LV et al.; H97N, H95N, and Y229F mutants of L-ribulose-5-phosphate 4-epimerase had 10, 1, and 0.1%, respectively, of the activity of the wild-type (WT) enzyme when activated by Zn(2+), the physiological activator . Co(2+) and Mn(2+) replaced Zn(2+) in Y229F and WT enzymes, although less effectively with the His mutants, while Mg(2+) was a poorly bound, weak activator . None of the other eight tyrosines mutated to phenylalanine caused a major loss of activity . The near-UV CD spectra of all enzymes were nearly identical in the absence of metal ions and substrate, and addition of substrate without metal ion showed no effect . When both substrate and Zn(2+) were present, however, the positive band at 266 nm increased while the negative one at 290 nm decreased in ellipticity . The changes for the WT and Y229F enzymes were greater than for the two His mutants . With Co(2+) as the metal ion, the CD and absorption spectra in the visible region were different, showing little ellipticity in the absence of substrate and a weak absorption band at 508 nm . With substrate present, however, an intense absorption band at 555 nm (epsilon = 150-175) with a negative molar ellipticity approaching 2000 deg cm(2) dmol(-1) appears with WT and Y229F enzymes . With the His mutants, the changes induced by substrate were smaller, with negative ellipticity only half as great . The WT, Y229F, H95N, and H97N enzymes all catalyze a slow aldol condensation of dihydroxyacetone and glycolaldehyde phosphate with an initial k(cat) of 1.6 x 10(-3) s(-1) . The initial rate slowed most rapidly with WT and H97N enzymes, which have the highest affinity for the ketopentose phosphates formed in the condensation . The EPR spectrum of enzyme with Mn(2+) exhibited a drastic decrease upon substrate addition, and by using H(2)(17)O, it was determined that there were three waters in the coordination sphere of Mn(2+) in the absence of substrate . These data suggest that (1) the substrate coordinates to the enzyme-bound metal ion, (2) His95 and His97 are likely metal ion ligands, and (3) Tyr229 is not a metal ion ligand, but may play another role in catalysis, possibly as an acid-base catalyst. Biochemistry, 2000 Apr 25, 39(16), 4808 - 20 13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase; Lee LV et al.; On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4 . With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively . H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2 . 69 and 1.99% at C-4, respectively . Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N . At C-4, the corresponding values were 9.6, 14, and 19% . These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6 . The Y229 mutant shows only the internal commitment of 0.6 . The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket . The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24 . The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm. Biochemistry, 2000 Apr 25, 39(16), 4722 - 8 Probing the catalytic mechanism of prephenate dehydratase by site-directed mutagenesis of the Escherichia coli P-protein dehydratase domain; Zhang S et al.; The Escherichia coli bifunctional P-protein, which plays a central role in L-phenylalanine (Phe) biosynthesis, contains distinct chorismate mutase (CM) and prephenate dehydratase (PDT) domains as well as a regulatory (R) domain for feedback control by Phe . To elucidate the catalytic mechanism of PDT in the P-protein, 24 mutations of 15 conserved residues in the PDT domain were created, expressed in the pheA(-)E . coli strain NK6024, and studied for their effect on PDT activity . Fourteen mutant enzymes were purified to homogeneity, tested for feedback inhibition by Phe, and characterized by kinetic analysis and circular dichroism spectroscopy . Selected mutant enzymes were further studied by gel filtration, fluorescence emission, and microcalorimetry . In addition, a monofunctional PDT domain (PDT20, residues 101-285) was cloned and overexpressed in plasmid pET with expression levels up to 200-250 mg/L . PDT20 retained full PDT activity, lacked CM activity, and was insensitive to feedback inhibition by Phe . Four residues (T278, N160, Q215, and S208) were shown to be important for PDT catalysis . The values of k(cat)/K(m) for the S208A/C and T278S mutant enzymes were 100-fold lower, and 500-fold lower for the N160A and Q215A mutant enzymes than the wild-type (WT) protein . The T278A and T278V mutant enzymes displayed no measurable catalytic activity, yet bound both prephenate and a competitive inhibitor (S-DNBA) comparably to the WT protein . These data, taken together with the normal CD spectra of the mutant enzymes, strongly suggested that T278 was involved in the catalytic mechanism . To establish whether acidic residues were involved in catalysis, all the conserved Glu and Asp residues in the PDT domain were mutated to Ala . None of these mutations significantly reduced PDT activity, indicating that the acidic residues of the PDT domain are not directly involved in catalysis . However, two mutant enzymes (E159A and E232A) displayed higher levels of PDT activity (2.2- and 3.5-fold, respectively), which was due to enhanced substrate binding . For the double mutant enzyme (E159A-E232A), k(cat)/K(m) was ca . 7-fold higher than for the WT enzyme, while its K(m) was 4.6-fold lower. Biochemistry, 2000 Apr 25, 39(16), 4704 - 10 The role of enzyme isomerization in the native catalytic cycle of the ATP sulfurylase-GTPase system; Wei J et al.; ATP sulfurylase, from E . coli Kappa-12, is a GTPase.target complex that conformationally couples the free energies of GTP hydrolysis and activated sulfate (adenosine 5'-phosphosulfate, or APS) synthesis . Energy coupling is achieved by an allosterically driven isomerization that switches on and off chemistry at specific points in the catalytic cycle . This coupling mechanism is derived from the results of model studies using analogue complexes that mimic different stages of the native catalytic cycle . The current investigation extends the analogue studies to the native catalytic cycle . Isomerization is monitored using the fluorescent, guanine nucleotide analogues mGMPPNP (3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-{beta, gamma-imido}triphosphate) and mGTP {3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-triphosphate} . The isomerization is shown to be initiated by an allosteric interaction that requires the simultaneous occupancy of all three substrate-binding sites . Stopped-flow fluorescence and single-turnover studies were used to define and quantitate the isomerization mechanism, and to show that the isomerization precedes and rate-limits both GTP hydrolysis and APS synthesis . These findings are incorporated into a model of the energy-coupling mechanism. Biochemistry, 2000 Apr 25, 39(16), 4640 - 8 Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit; Guittet O et al.; Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase . The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity . This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues . In this report, nitrated residues in the E . coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing . Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit . The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da . Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein . A mean of two nitrotyrosines per E . coli protein R2 dimer were obtained at 400 microM peroxynitrite . Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122 . Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility . Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed . However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E . coli protein R2. Biochemistry, 2000 Apr 25, 39(16), 4630 - 9 New reactions in the crotonase superfamily: structure of methylmalonyl CoA decarboxylase from Escherichia coli; Benning MM et al.; The molecular structure of methylmalonyl CoA decarboxylase (MMCD), a newly defined member of the crotonase superfamily encoded by the Escherichia coli genome, has been solved by X-ray crystallographic analyses to a resolution of 1.85 A for the unliganded form and to a resolution of 2.7 A for a complex with an inert thioether analogue of methylmalonyl CoA . Like two other structurally characterized members of the crotonase superfamily (crotonase and dienoyl CoA isomerase), MMCD is a hexamer (dimer of trimers) with each polypeptide chain composed of two structural motifs . The larger N-terminal domain contains the active site while the smaller C-terminal motif is alpha-helical and involved primarily in trimerization . Unlike the other members of the crotonase superfamily, however, the C-terminal motif is folded back onto the N-terminal domain such that each active site is wholly contained within a single subunit . The carboxylate group of the thioether analogue of methylmalonyl CoA is hydrogen bonded to the peptidic NH group of Gly 110 and the imidazole ring of His 66 . From modeling studies, it appears that Tyr 140 is positioned within the active site to participate in the decarboxylation reaction by orienting the carboxylate group of methylmalonyl CoA so that it is orthogonal to the plane of the thioester carbonyl group . Surprisingly, while the active site of MMCD contains Glu 113, which is homologous to the general acid/base Glu 144 in the active site of crotonase, its carboxylate side chain is hydrogen bonded to Arg 86, suggesting that it is not directly involved in catalysis . The new constellation of putative functional groups observed in the active site of MMCD underscores the diversity of function in this superfamily. Biochemistry, 2000 Apr 25, 39(16), 4622 - 9 Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli; Haller T et al.; The Escherichia coli genome encodes seven paralogues of the crotonase (enoyl CoA hydratase) superfamily . Four of these have unknown or uncertain functions; their existence was unknown prior to the completion of the E . coli genome sequencing project . The gene encoding one of these, YgfG, is located in a four-gene operon that encodes homologues of methylmalonyl CoA mutases (Sbm) and acyl CoA transferases (YgfH) as well as a putative protein kinase (YgfD/ArgK) . We have determined that YgfG is methylmalonyl CoA decarboxylase, YgfH is propionyl CoA:succinate CoA transferase, and Sbm is methylmalonyl CoA mutase . These reactions are sufficient to form a metabolic cycle by which E . coli can catalyze the decarboxylation of succinate to propionate, although the metabolic context of this cycle is unknown . The identification of YgfG as methylmalonyl CoA decarboxylase expands the range of reactions catalyzed by members of the crotonase superfamily. Biochemistry, 2000 Apr 25, 39(16), 4590 - 602 Evolution of enzymatic activities in the enolase superfamily: crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli; Gulick AM et al.; D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydration of both D-glucarate and L-idarate as well as their interconversion via epimerization . GlucD is a member of the mandelate racemase (MR) subgroup of the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion substrate . Alignment of the sequence of GlucD with that of MR reveals a conserved Lys-X-Lys motif and a His-Asp dyad homologous to the S- and R-specific bases in the active site of MR . Crystals of GlucD have been obtained into which the substrate D-glucarate and two competitive inhibitors, 4-deoxy-D-glucarate and xylarohydroxamate, could be diffused; D-glucarate is converted to the dehydration product, 5-keto-4-deoxy-D-glucarate (KDG) . The structures of these complexes have been determined and reveal the identities of the ligands for the required Mg(2+) (Asp(235), Glu(266), and Asn(289)) as well as confirm the expected presence of Lys(207) and His(339), the catalytic bases that are properly positioned to abstract the proton from C5 of L-idarate and D-glucarate, respectively . Surprisingly, the C6 carboxylate group of KDG is a bidentate ligand to the Mg(2+), with the resulting geometry of the bound KDG suggesting that stereochemical roles of Lys(207) and His(339) are reversed from the predictions made on the basis of the established structure-function relationships for the MR-catalyzed reaction . The catalytic roles of these residues have been examined by characterization of mutant enzymes, although we were unable to use these to demonstrate the catalytic independence of Lys(207) and His(339) as was possible for the homologous Lys(166) and His(297) in the MR-catalyzed reaction. J Gen Virol, 2000 May, 81(Pt 5), 1335 - 45 Enzymatic properties of hepatitis C virus NS3-associated helicase; Paolini C et al.; The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion . In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed in Escherichia coli and purified to homogeneity by conventional chromatography . Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates . On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3' to 5' direction . In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA . Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem-loop RNA structure (SL I) within the 3'-terminal 46 bases of the viral genome . Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule. J Histochem Cytochem, 2000 May, 48(5), 685 - 93 Immunohistochemical localization of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in rat tissues; Kotera J et al.; We raised a polyclonal antibody against maltose binding protein fusion human cGMP-binding, cGMP-specific phosphodiesterase (PDE5) produced in E . coli . This antibody immunoreacted specifically with recombinant human and rat PDE5 proteins expressed in transfected COS-7 cells and with a native form of PDE5 in extracts of rat platelets, lung, and cerebellum . Immunohistochemical analysis showed that the anti-PDE5 antibody detected immunoactive materials in Purkinje cell layers of the cerebellum, proximal renal tubules, collecting renal ducts, and epithelial cells of pancreatic ducts in rats . Reverse transcriptase-polymerase chain reaction analysis demonstrated that PDE5 transcripts are also present in rat cerebellum, kidney, and pancreas . Here we described a cell-specific localization of PDE5 in various rat tissues, suggesting the possibility of the presence of a cGMP/PDE5 pathway in these tissues. Infect Immun, 2000 May, 68(5), 2954 - 61 Characterization of heat, oxidative, and acid stress responses in Brucella melitensis; Teixeira-Gomes AP et al.; Brucella melitensis is a facultative intracellular pathogen which is able to survive and replicate within phagocytic cells . Therefore, it has to adapt to a range of different hostile environments . In order to understand the mechanisms of intracellular survival employed by virulent B . melitensis 16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses by two-dimensional (2-D) polyacrylamide gel electrophoresis was used . Depending on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis . On the basis of N-terminal sequence analysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified . Some of them were previously reported for Brucella, such as BvrR, DnaK, GroEL, and Cu-Zn superoxide dismutase (SOD) . Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydrogenase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 component beta subunit . Results indicated that B . melitensis could bring specific regulatory mechanisms into play in response to stress conditions . For example, the ribosome releasing factor in B . melitensis appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for Escherichia coli, was strongly down-regulated in B . melitensis in response to heat stress . Some of the identified proteins and their potential specific regulation could be required for the adaptation of B . melitensis to environmental stresses encountered in phagocytic cells and possibly for bacterial virulence. Infect Immun, 2000 May, 68(5), 2791 - 6 mkp-1 encoding mitogen-activated protein kinase phosphatase 1, a verotoxin 1 responsive gene, detected by differential display reverse transcription-PCR in Caco-2 cells; Kojima S et al.; The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing Escherichia coli is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release . Four differentially expressed genes, including mkp-1 (encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells . Northern blot analysis showed the induction of mkp-1 mRNA 6 h after VT1 stimulation . Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced mkp-1 mRNA, but mkp-1 mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor . Therefore, we concluded that the A subunit of VT1 was essential for mkp-1 induction . Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin . Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1 . This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins. Infect Immun, 2000 May, 68(5), 2775 - 82 Parenteral adjuvant activities of Escherichia coli heat-labile toxin and its B subunit for immunization of mice against gastric Helicobacter pylori infection; Weltzin R et al.; The heat-labile toxin (LT) of Escherichia coli is a potent mucosal adjuvant that has been used to induce protective immunity against Helicobacter felis and Helicobacter pylori infection in mice . We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered with H . pylori urease antigen via the parenteral route . Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge with H . pylori . Control mice were immunized orally with urease plus LT, a regimen shown previously to protect against H . pylori gastric infection . Parenteral immunization using either LT or LTB as adjuvant protected mice against H . pylori challenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant . LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization . A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum . Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used . Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a . Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice . These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice against H . pylori infection. Infect Immun, 2000 May, 68(5), 2766 - 74 Identification of a gene within a pathogenicity island of enterotoxigenic Escherichia coli H10407 required for maximal secretion of the heat-labile enterotoxin; Fleckenstein JM et al.; Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens . ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease . These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis . Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E . coli pathogenicity islands . Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia . The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses . An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection . Although previous studies have suggested that E . coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island. J Physiol Pharmacol, 2000 Mar, 51(1), 85 - 102 Protective role of endogenous nitric oxide (NO) in lipopolysaccharide--induced pancreatic damage (a new experimental model of acute pancreatitis); Jaworek J et al.; Lipopolysaccharide (LPS) derived from the bacterial cell wall activates the inflammatory response in the tissue but the role of LPS in the pathogenesis of pancreatic damage and in the activation of NO system in the pancreas has not been fully explained . The aim of this study was to investigate the effect of repeated administration of LPS to the rats on the integrity of the pancreas, on the ability of isolated pancreatic acini to secrete the amylase and on the plasma level of tumor necrosis factor alpha (TNFalpha) . The role of NO in the pancreatic resistance to the damage was assessed in animals subjected to repeated administration of LPS . To induce pancreatic damage one group of rats received intraperitoneal (i.p.) injection of LPS (from E . coli) every day during 5 consecutive days (10 mg/kg--day) . Another groups of animals were given N(G)-nitro-L-arginine (L-NNA), an inhibitor of NO synthase (NOS) (20 mg/kg i.p.) alone or in combination with L-arginine (100 mg/kg i.p.), 30 min prior to each LPS injection . Plasma level of TNFalpha was determined by ELISA kit . Repeated administration of LPS produced mild pancreatic inflammation that was most pronounced at day 5 of LPS treatment and manifested as edema, neutrophil infiltration and hemorrhage of the pancreas . The survival rate after 5 days treatment with LPS was 87.5% . Pancreatic weight, plasma levels of TNFalpha and amylase, pancreatic blood flow (PBF) and NO generation by pancreatic acini were markedly increased in rats subjected to repeated administration of LPS whereas the amylase response of isolated pancreatic acini to pancreatic secretagogues was significantly attenuated . Suppression of NOS by L-NNA resulted in a dramatic increase in the mortality of the animals reaching 50% and significantly increased inflammatory changes in the pancreatic tissue, decreased PBF, abolished the ability of pancreatic acini to release NO and to secrete amylase . Pancreatic weight and plasma levels of amylase and TNFalpha significantly increased in the group of rats treated with combination of LPS+L-NNA as compared to the animals received LPS alone . Addition of L-arginine to L-NNA+LPS administration reversed all harmful effects produced by L-NNA in the pancreas . We conclude that repeated administration of high doses of bacterial LPS to the rats could induce pancreatic tissue damage by itself, however, it is not able to produce severe pancreatitis . Suppression of NO generation significantly aggravates the pancreatic lesion produced by LPS leading to the dramatic mortality in treated rats . The rise of plasma level of TNFalpha corresponds to the severity of pancreatic inflammation. Hybridoma, 2000 Feb, 19(1), 1 - 13 Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus; Long MC et al.; A novel recombinant single-chain fragment variable (scFv) antibody against Western equine encephalitis virus (WEE) was constructed and characterized . Using antibody phage display technology, a scFv was generated from the WEE specific hybridoma, 10B5 E7E2 . The scFv was fused to a human heavy chain IgG1 constant region (CH1-CH3) and contained an intact 6 His tag and enterokinase recognition site (RS10B5huFc) . The RS10B5huFc antibody was expressed in E . coli and purified by affinity chromatography as a 70-kDa protein . The RS10B5huFc antibody was functional in binding to WEE antigen in indirect enzyme-linked immunosorbent assays (ELISAs) . Furthermore, the RS10B5huFc antibody was purified in proper conformation and formed multimers . The addition of the human heavy chain to the scFv replaced effector functions of the mouse antibody . The Fc domain was capable of binding to protein G and human complement . The above properties of the RS10B5huFc antibody make it an excellent candidate for immunodetection and immunotherapy studies. Electrophoresis, 2000 Mar, 21(5), 993 - 1000 A comparison of three commercially available isoelectric focusing units for proteome analysis: the multiphor, the IPGphor and the protean IEF cell; Choe LH et al.; We tested and compared three different commercially available instruments for isoelectric focusing for proteome analysis by two-dimensional protein electrophoresis . These instruments, the Multiphor, the IPGphor, and the Protean IEF cell, were used with 18 cm immobilized pH gradient strips and run under various conditions . The total number of spots and features was obtained by Melanie software (Bio-Rad Laboratories) and separately by visual inspection . The Multiphor consistently resulted in the highest number of spots detected per gel independent of sample type, immobilized pH gradient (IPG) and method to calculate the number of spots . The Protean IEF cell had the next highest number of spots detected per gel . In the experiments performed, the IPGphor afforded good reproducibility in the total number of Melanie-detected spots from gel to gel while the Protean IEF cell offered better reproducibility in the total number of manually detected spots from gel to gel . Among gels run with the different instruments, differences in the quality of the ammoniacal silver stain were also observed . A measure of quantitative reproducibility suggests that the Protean IEF cell, which was the easiest instrument to use, performs better than the other instruments, although all three instruments had demonstrated good quantitative reproducibility in the experiments performed. J Chromatogr A, 2000 Mar 31, 874(1), 27 - 43 Immobilised metal affinity chromatography of beta-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption; Clemmitt RH et al.; The development of an expanded bed process for the direct extraction and partial purification of beta-galactosidase from unclarified Escherichia coli homogenates using its natural affinity for metal loaded STREAMLINE Chelating is described . Small packed beds were used to determine the effect of chelated metal ion (Cu2+, Ni2+, Co2+ or Zn2+), loading pH and ionic strength on the selective binding capacity, and recovery of beta-galactosidase from clarified homogenates . An elution protocol was developed using the competitive displacer, imidazole, to recover beta-galactosidase in 87% yield and 3.4-fold purification . These results were then used to develop a separation for the recovery of beta-galactosidase from unclarified homogenates in a 2.5-cm diameter expanded bed . Although Ni2+ loaded STREAMLINE Chelating had a 5% dynamic capacity for beta-galactosidase of just 118 U ml(-1) (0.39 mg ml(-1)), the low capacity was thought to be due to the large size of the target (464,000) relative to the exclusion limit of the macroporous adsorbent . Despite this low capacity, Ni2 STREAMLINE Chelating was used successfully to recover beta-galactosidase from an unclarified homogenate in 86.4% yield and at 5.95-fold purification . The degree of purification relative to a commercial standard, as assessed using the purification factor and sodium dodecyl sulphate-polyacrylamide gel electrophoresis was high suggesting that this pseudo-affinity procedure compared favourably with alternative methods. Mutat Res, 2000 Apr, 462(2-3), 71 - 82 Unmasking a killer: DNA O(6)-methylguanine and the cytotoxicity of methylating agents; Bignami M et al.; Methylating agents are potent carcinogens that are mutagenic and cytotoxic towards bacteria and mammalian cells . Their effects can be ascribed to an ability to modify DNA covalently . Pioneering studies of the chemical reactivity of methylating agents towards DNA components and their effectiveness as animal carcinogens identified O(6)-methylguanine (O(6)meG) as a potentially important DNA lesion . Subsequent analysis of the effects of methylating carcinogens in bacteria and cultured mammalian cells - including the discovery of the inducible adaptive response to alkylating agents in Escherichia coli - have defined the contributions of O(6)meG and other methylated DNA bases to the biological effects of these chemicals . More recently, the role of O(6)meG in killing mammalian cells has been revealed by the lethal interaction between persistent DNA O(6)meG and the mismatch repair pathway . Here, we briefly review the results which led to the identification of the biological consequences of persistent DNA O(6)meG . We consider the possible consequences for a human cell of chronic exposure to low levels of a methylating agent . Such exposure may increase the probability that the cell's mismatch repair pathway becomes inactive . Loss of mismatch repair predisposes the cell to mutation induction, not only through uncorrected replication errors but also by methylating agents and other mutagens. Gene, 2000 Apr 4, 246(1-2), 321 - 30 PCR-mediated gene replacement in Escherichia coli; Murphy KC et al.; The hyper-recombinogenic properties of an E . coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli . Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCDDelta::red substitution . This technique allows for gene replacement in E . coli without prior cloning of the gene of interest . In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates . In other experiments, electroporation of recBCDDelta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation . The placement of lambda red and gam at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyper-rec as one containing the lambda red for recBCD substitution . The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E . coli . Three of these replacements were performed without prior cloning of the genes. Gene, 2000 Apr 4, 246(1-2), 255 - 64 Identification and recombinant expression of glyceraldehyde-3-phosphate dehydrogenase of Plasmodium falciparum; Daubenberger CA et al.; The gene coding for the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was isolated from Plasmodium falciparum . The gene contains 1 intron and the A+T content is characteristic for the codon usage of P . falciparum . The predicted open reading frame codes for 337 amino acids (36651Da) and is 63.5% identical to the human erythrocytic GAPDH . GAPDH sequences from several field isolates of P . falciparum displayed 100% conservation . Phylogenetic analysis supports the hypothesis that dinoflagellates and Plasmodium are closely related . The protein encoded by the pfGAPDH was expressed recombinantly in Escherichia coli and exhibited enzymatic activity with NAD(+) but not with NADP(+) as cofactor . Antiserum raised against the recombinantly expressed enzyme detected specifically all developmental stages of cultured P . falciparum blood-stage parasites. Gene, 2000 Apr 4, 246(1-2), 151 - 5 Candida glabrata shuttle vectors suitable for translational fusions to lacZ and use of beta-galactosidase as a reporter of gene expression; El Barkani A et al.; The functionality of beta-galactosidase encoded by the E . coli lacZ gene as a reporter of gene expression in C . glabrata was investigated . C . glabrata/E . coli shuttle vectors were constructed, containing both a C . glabrata CEN-ARS cassette, to allow regular segregation and episomal replication of the plasmids, and the lacZ coding sequence of E . coli . The functionality of beta-galactosidase in C . glabrata was verified by inserting the promoter and the 5' coding region of the HIS3 gene from C . glabrata directionally upstream of the lacZ gene . By fusing the promoter of the copper-controlled MTII gene to the lacZ reporter, we showed that beta-galactosidase activity can be differentially induced in C . glabrata . beta-galactosidase reporter activities were detected qualitatively by an indirect filter assay and quantitatively from permeabilized cells. FEBS Lett, 2000 Apr 14, 471(2-3), 211 - 4 GroES co-chaperonin small-angle X-ray scattering study shows ring orifice increase in solution; Timchenko AA et al.; GroES consists of seven identical 10 kDa subunits and is involved in assisting protein folding as the partner of another oligomeric protein, the GroEL chaperonin . Here we studied the GroES structure in solution using small-angle X-ray scattering (SAXS) . The SAXS pattern, calculated for the GroES crystal structure, was found to be different from the experimental one measured in solution . The synchronic shift in the radial direction and some turning of the protein subunits eliminate the difference and result in the increase of the hole diameter in the GroES ring-like structure from 8 A in the crystal to 21 A in solution. FEBS Lett, 2000 Apr 14, 471(2-3), 173 - 6 Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration; Simon H et al.; The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated . The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration . As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site . The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents. J Exp Zool, 2000 May 1, 286(6), 656 - 65 cDNA cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle; Kimura A et al.; A cDNA for rat prolyl oligopeptidase was cloned which contained an open reading frame of 2,130 nucleotides encoding a protein of 710 amino acids . The deduced amino acid sequence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% to bacterial prolyl oligopeptidases . The recombinant prolyl oligopeptidase generated in E . coli was purified and its properties were examined . The substrate specificity and the susceptibility to proteinase inhibitors were similar to those of the native enzyme . Northern blot analysis showed wide expression of the prolyl oligopeptidase gene . Using ovaries from hormone-treated rats, it was found that both the mRNA expression and enzyme activity increased in the luteal phase . These findings suggest the involvement of prolyl oligopeptidase in events associated with corpus luteum formation and/or luteal regression. J Biol Chem, 2000 Apr 21, 275(16), 12261 - 5 Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme; Arnold DA et al.; Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination . Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion . Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme . However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele . Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo. J Biol Chem, 2000 Apr 21, 275(16), 12214 - 22 Transcriptional regulation of the divergent paa catabolic operons for phenylacetic acid degradation in Escherichia coli; Ferrandez A et al.; The expression of the divergently transcribed paaZ and paaABCDEFGHIJK catabolic operons, which are responsible for phenylacetic acid (PA) degradation in Escherichia coli, is driven by the Pz and Pa promoters, respectively . To study the transcriptional regulation of the inducible paa catabolic genes, genetic and biochemical approaches were used . Gel retardation assays showing that the PaaX regulator binds specifically to the Pa and Pz promoters were complemented with in vivo experiments that indicated a PaaX-mediated repression effect on the expression of Pa-lacZ and Pz-lacZ reporter fusions . The region within the Pa and Pz promoters that is protected by the PaaX repressor in DNase I footprinting assays contains a conserved 15-base pair imperfect palindromic sequence motif that was shown, through mutational analysis, to be indispensable for PaaX binding and repression . PA-coenzyme A (PA-CoA), but not PA, specifically inhibited binding of PaaX to the target sequences, thus confirming the first intermediate of the pathway as the true inducer and PaaX as the only bacterial regulatory protein described so far that responds to an aryl-CoA compound . Superimposed in the specific PaaX-mediated regulation is transcriptional activation by the cAMP receptor protein and the integration host factor protein . These global regulators may adjust the transcriptional output from Pa and Pz promoters to the overall growth status of the cell. J Biol Chem, 2000 Apr 21, 275(16), 12009 - 16 The yeast mitochondrial citrate transport protein . Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway; Kaplan RS et al.; The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine . A cysteine-less CTP retaining wild-type functional properties served as the starting template . The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system . The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function . These rate constants varied by seven orders of magnitude . With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193 . Based on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix . Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192 . We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. J Biol Chem, 2000 Apr 21, 275(16), 11951 - 6 Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression; Meibom KL et al.; At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes . Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes . Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP is responsible for the transcriptional inactivity of the complexes. J Biol Chem, 2000 Apr 21, 275(16), 11915 - 20 Filling the gap in vitamin A research . Molecular identification of an enzyme cleaving beta-carotene to retinal; von Lintig J et al.; Vitamin A and its derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation with important clinical implications . It has been known for 50 years that the key step in the formation of vitamin A is the oxidative cleavage of beta-carotene; however, this enzymatic step has resisted molecular analysis . A novel approach enabled us to clone and identify a beta-carotene dioxygenase from Drosophila melanogaster, expressing it into the background of a beta-carotene (provitamin A)-synthesizing and -accumulating Escherichia coli strain . The carotene-cleaving enzyme, identified here for the first time on the molecular level, is the basis of the numerous branches of vitamin A action and links plant and animal carotene metabolism. J Biol Chem, 2000 Apr 21, 275(16), 11865 - 73 Clustered DNA damage, influence on damage excision by XRS5 nuclear extracts and Escherichia coli Nth and Fpg proteins; David-Cordonnier MH et al.; Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity . Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage . These proteins were tested for their efficiency to induce a single-strand break on a (32)P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2'deoxyguanine, or 7, 8-dihydro-8-oxo-2'deoxyadenine at positions 1, 3, or 5 base pairs 5' or 3' to either an AP site or DHT on the labeled strand . DHT excision is much more affected than cleavage of an AP site by the presence of other damage . The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg . Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages. J Biol Chem, 2000 Apr 21, 275(16), 11758 - 64 Identification of the pore-forming region of the outer chloroplast envelope protein OEP16; Steinkamp T et al.; The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids . The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants . Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli . The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein . Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel . The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel . Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93. J Biol Chem, 2000 Apr 21, 275(16), 11672 - 7 A distinct seven-residue trigger sequence is indispensable for proper coiled-coil formation of the human macrophage scavenger receptor oligomerization domain; Frank S et al.; We have recently identified a distinct 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils but not in trimers, tetramers, or pentamers . This coiled-coil trigger pattern was demonstrated to be indispensable for the assembly of the oligomerization domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum and the leucine zipper domain of the yeast transcriptional activator GCN4 . With the aim to extend our knowledge on trigger sequences we have investigated the human macrophage scavenger receptor type A oligomerization domain as a representative of three-stranded coiled coils . We prepared a variety of recombinant N- and C-terminal deletion mutants from the full-length oligomerization domain by heterologous gene expression in Escherichia coli and assessed their ability to form trimeric coiled-coil structures by circular dichroism spectroscopy and analytical ultracentrifugation . Deletion mapping identified a distinct seven-residue sequence that was absolutely required for proper coiled-coil formation, supporting our previous results that heptad repeats alone are not sufficient for oligomerization . The finding that all fragments containing this particular sequence exhibited similar thermal stabilities indicates primarily a stabilizing function of the coiled-coil trigger . Based on sequence similarity, we suggest that functionally related sites are present in other three-stranded coiled-coil proteins. J Biol Chem, 2000 Apr 21, 275(16), 11610 - 7 Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids; Powell KA et al.; Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC) . Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization . An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid . Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM . Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone . Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin . Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase . However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain . Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals. J Biol Chem, 2000 Apr 21, 275(16), 11591 - 6 The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli; Stanley NR et al.; In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif . The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI . Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue . Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair . The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain . Unexpectedly, the consensus lysine residue was found to retard Tat transport . These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues . Tat signal peptides characteristically have positively charged carboxyl-terminal regions . However, changing the sign of this charge does not affect export of SufI. J Biol Chem, 2000 Apr 21, 275(16), 11576 - 84 Sequence-specific interaction between the disintegrin domain of mouse ADAM 2 (fertilin beta) and murine eggs . Role of the alpha(6) integrin subunit; Bigler D et al.; Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins . Mouse ADAM 2 (mADAM 2; fertilin beta) is a sperm surface protein involved in murine fertilization . We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria . The protein produced in insect cells (baculo D+C) contained a signal sequence followed by the disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells . A bacterial construct containing the disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera . Baculo D+C, as well as the D domain of the bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs . Using concentrations in the range of 1 to 5 microM, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (approximately 75 versus approximately 55% maximal inhibition) . Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine . Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-alpha(6) monoclonal antibody, GoH3, but not by a nonfunction-blocking anti-alpha(6) monoclonal antibody . Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2. J Biol Chem, 2000 Apr 21, 275(16), 11541 - 4 Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein; Jhee KH et al.; Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease) . We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase . The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase . The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism . The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm . The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction . Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function . The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes . The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase. J Biol Chem, 2000 Jun 30, 275(26), 19482 - 9 Butadiene-induced intrastrand DNA cross-links: a possible role in deletion mutagenesis; Carmical JR et al.; To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon . The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli . Both stereoisomeric forms (R, R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic . Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E . coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links . The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E . coli UvrABC complex . UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system. J Biol Chem, 2000 Jul 14, 275(28), 21349 - 54 X-ray scattering studies of Methylophilus methylotrophus (sp . W3A1) electron-transferring flavoprotein . Evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase; Jones M et al.; Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp . W(3)A(1)) . Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states . Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution . These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible "hinge" sequences that are unique to M . methylotrophus ETF . The x-ray scattering data are consistent with previous models concerning the interaction of M . methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase . Our data reveal that an "induced fit" mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process. J Biol Inorg Chem, 2000 Feb, 5(1), 67 - 74 Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry; Cowan JA et al.; Crystallographic studies of the Mn(2+)-doped RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have revealed two bound Mn2+ separated by approximately 4A and surrounded by a cluster of four conserved carboxylates . Escherichia coli RNase H is structurally similar to the RNase H domain of HIV-1 RT, but requires one divalent metal cation for its activity, implying either that the HIV-1 RT RNase H domain contrasts in its ability to bind two divalent metal ions, or that the crystallographic data reflect specific use of Mn2+ and/ or the doping technique employed . Metal binding stoichiometry has been determined for Mn2+ and the biologically more relevant Mg2+ cation by solution calorimetric studies of native and recombinant p66/p51 HIV-1 RT . Three Mn2+ ions bind to HIV-1 RT apo-enzyme: one at the DNA polymerase and two at the RNase H catalytic center, the latter being consistent with crystallographic results . However, only one Mg2+ ion is bound in the RNase H catalytic center . Several mechanistic implications arise from these results, including the possibility of mutually exclusive Mg2+ binding sites that might be occupied according to the specific reaction being catalyzed by the multifunctional RNase H domain . The occurrence of distinct binding stoichiometries for Mg2+ and Mn2+ to multifunctional enzymes has previously been reported. Anticancer Drug Des, 1999 Oct, 14(5), 411 - 20 A similarity model for the human angiogenic factor, thymidine phosphorylase/platelet derived-endothelial cell growth factor; Cole C et al.; Thymidine phosphorylase (EC 2.4.2.4), identical to the angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), is up-regulated in several tumour types . A similarity model of human thymidine phosphorylase was built, based on the crystal structure of the Escherichia coli enzyme . The high residue conservation between the two enzyme sources (39% sequence identity and 53% sequence similarity) aided model building . The human model was very similar to the E . coli enzyme's crystal structure, with the main tertiary structure difference being the destruction of helix 15 in E . coli by the presence of a loop in the human model . The model was used to rationalize the nature of the binding of the substrates thymine and thymidine, and of known inhibitors using a quantitative docking algorithm . Ab initio calculations on the nM inhibitor 5-chloro-6-(1-(2-iminopyrrolidinyl)methyl)uracil hydrochloride gave its conformation and distribution of charge . Subsequent quantitative docking studies have led to the suggestion, for the first time, that this inhibitor behaves as an oxycarbenium ion transition-state analogue, explaining its strong reported inhibition. Vestn Ross Akad Med Nauk, 2000, (3), 49 - 53 {Carbohydrate transport systems in Escherichia coli and regulation of catabolism}; Gershanovich VN; The mechanism of carbohydrate uptake in E . coli with involvement of the phosphoenol pyruvate-dependent phosphotransferase system (PTS) is dealt with . The genetic structure of the glucose transport system and fructose operon is given in detail . How the products of these systems can affect the total bacterial cellular catabolism dependent of complex of cyclic 3'5'-monophosphate + protein regulator of the above nucleotide is shown . Some section is devoted to the bacterial transport of beta-glycosides with the participation of Po-independent terminator and to the role of PTS as a highly sensitive sensory system associated with carbohydrate catabolism. Domest Anim Endocrinol, 2000 Feb, 18(2), 187 - 97 Alterations of growth hormone, cortisol, luteinizing hormone, and insulin concentrations in early-postnatal calves affected with diarrhea; Bruckmann A et al.; The aim of the study was to investigate the influence of diarrheic infections during the early postnatal phase of calves on the concentrations of hormones controlling reproduction and metabolism . Blood samples were collected from 20 male and female calves via jugular vein catheters every 15 min for 6 hr at Days 3, 9, and 21 of life . The animals were classified into three groups . Group 1 (controls): healthy calves (n = 9) . Group 2: calves affected with diarrhea at Day 9 (n = 7) . Group 3: calves with diarrhea at Days 3 and 9 (n = 4) . Infections occurred spontaneously and were mainly due to E . coli infections . All affected calves had recovered at Day 21 . Mean GH concentrations in the calves in Groups 2 and 3 compared to control calves had increased by Day 3 (P<0.01; P<0.001) . Cortisol levels of calves in all groups were highest at Day 3 and decreased thereafter (P<0.001) . Cortisol concentrations were lower at Day 3 in animals in Groups 2 (P<0.001) and 3 (P<0.05) than in controls . Pulsatile LH release was detectable at Days 9 and 21 only in healthy calves . Insulin increased at Day 9 during diarrhea . The results indicate that cortisol concentrations decreased whereas GH concentrations were increased before diarrhea was observed . The onset of pulsatile LH release was delayed in diarrheic calves . It is concluded that diarrhea exerts effects upon the release of reproductive and metabolic hormones in early postnatal calves. J Biol Chem, 2000 Jun 23, 275(25), 19098 - 105 Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain; Cruz MA et al.; The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury . It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb) . The GPIb-binding site within vWF has been localized to the vWF-A1 domain . Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R . (1998) J . Biol . Chem . 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site . We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations . The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination . Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site . One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here. J Biol Chem, 2000 Jun 16, 275(24), 18557 - 65 cDNA cloning, expression, and functional characterization of PI31, a proline-rich inhibitor of the proteasome; McCutchen-Maloney SL et al.; The primary structure of PI31, a protein inhibitor of the 20 S proteasome, was deduced by cDNA cloning and sequencing . The human protein has a calculated molecular weight of 29,792, a value in excellent accord with 31,000, as estimated by SDS-polyacrylamide gel electrophoresis for purified bovine PI31, and is not similar to any other protein in current data bases . PI31 is a proline-rich protein, particularly within its carboxyl-terminal half where 26% of the amino acids are proline . Wild-type PI31 and various truncation mutants were expressed in Escherichia coli and purified to homogeneity . Recombinant wild-type PI31 displayed structural and functional properties similar to those of PI31 purified from bovine red blood cells and inhibited the hydrolysis of protein and peptide substrates by the 20 S proteasome . Analysis of truncation mutants demonstrated that proteasome inhibition was conferred by the carboxyl-terminal proline-rich domain of PI31, which appears to have an extended secondary structure . Inhibition of the 20 S proteasome by PI31 involved formation a proteasome-PI31 complex . In addition to its direct inhibition of the 20 S proteasome, PI31 inhibited the activation of the proteasome by each of two proteasome regulatory proteins, PA700 and PA28 . These results suggest that PI31 plays an important role in control of proteasome function, including that in ubiquitin-dependent pathways of protein degradation. J Biol Chem, 2000 Jun 30, 275(26), 19461 - 8 Functional mapping of the GAGA factor assigns its transcriptional activity to the C-terminal glutamine-rich domain; Vaquero A et al.; GAGA is a nuclear protein encoded by the Trithorax-like gene in Drosophila that is expressed in at least two isoforms generated by alternative splicing . By means of its specific interaction with DNA, GAGA has been involved in several nuclear transactions including regulation of gene expression . Here we have studied the GAGA(519) isoform as a transcription factor . In vitro, the transactivation domain has been assigned to the 93 C-terminal residues that correspond to a glutamine-rich domain (Q-domain) . It presents an internal modular structure and acts independently of the rest of the protein . In vivo, in Drosophila SL2 cells, Q-domain can transactivate reporter genes either in the form of GAGA or Gal4BD-Q fusions, whereas a GAGA mutant deleted of the Q-domain cannot . Our results give support to the notion that GAGA can function as a transcription activating factor. J Biol Chem, 2000 Jun 23, 275(25), 18818 - 23 Ligand binding and structural properties of segments of GABAA receptor alpha 1 subunit overexpressed in Escherichia coli; Hang J et al.; The gamma-aminobutyric acid, type A (GABA(A)), receptor is the target for numerous therapeutic compounds . In the present study, the Gln(28)-Leu(296), Gln(28)-Arg(276), Gln(28)-Arg(248), and Gln(28)-Glu(165) (numbering of bovine precursor protein) segments of its alpha(1) subunit were overexpressed in Escherichia coli, along with Cys(166)-Leu(296) produced previously, for structural analysis by circular dichroism and ligand binding studies by fluorescence spectroscopy . Results showed that the protein segments were rich in beta-sheet structures . Binding of the fluorescent benzodiazepine Bodipy-FL Ro-1986 was evident from fluorescence resonance energy transfer and fluorescence anisotropy measurements . The binding affinity was in the micromolar range . The binding was attributable more to Cys(166)-Leu(296) than to Gln(28)-Glu(165) and was inhibited by known central benzodiazepine site ligands . Three point mutations, Y187A, T234A, and Y237A, were found to perturb protein secondary structures . Studies with the single Trp mutants W198Y and W273Y indicated that Trp(273) was closer to the binding site than Trp(198). J Biol Chem, 2000 Jun 2, 275(22), 16401 - 3 A common interface on histidine-containing phosphocarrier protein for interaction with its partner proteins; Wang G et al.; The bacterial phosphoenolpyruvate:sugar phosphotransferase system accomplishes both the transport and phosphorylation of sugars as well as the regulation of some cellular processes . An important component of this system is the histidine-containing phosphocarrier protein, HPr, which accepts a phosphoryl group from enzyme I, transfers a phosphoryl group to IIA proteins, and is an allosteric regulator of glycogen phosphorylase . Because the nature of the surface on HPr that interacts with this multiplicity of proteins from Escherichia coli was previously undefined, we investigated these interactions by nuclear magnetic resonance spectroscopy . The chemical shift changes of the backbone and side-chain amide (1)H and (15)N nuclei of uniformly (15)N-labeled HPr in the absence and presence of natural abundance glycogen phosphorylase, glucose-specific enzyme IIA, or the N-terminal domain of enzyme I have been determined . Mapping these chemical shift perturbations onto the three-dimensional structure of HPr permitted us to identify the binding surface(s) of HPr for interaction with these proteins . Here we show that the mapped interfaces on HPr are remarkably similar, indicating that HPr employs a similar surface in binding to its partners. J Mol Biol, 2000 Apr 28, 298(2), 273 - 82 High-resolution structure of the OmpA membrane domain; Pautsch A et al.; The membrane domain of OmpA consists of an eight-stranded all-next-neighbor antiparallel beta-barrel with short turns at the periplasmic barrel end and long flexible loops at the external end . The structure analysis has been extended from medium resolution to 1 . 65 A (1 A=0.1 nm), and the molecular model has been refined anisotropically to show oriented mobilities of the structural elements . The improved data allowed us to locate five further detergent molecules and 11 more water molecules . Moreover, the two large non-polar packing contacts have now been defined in detail . The analysis indicates that the beta-barrel constitutes a solid scaffold such that the long external loops need not contribute to stability . These loops are highly mobile and thus cause a major problem during the crystallization process . The beta-barrel was related to those of lipocalins . Two further crystal forms with exceptionally dense packing arrangements were established at medium resolution . J Mol Biol, 2000 Apr 28, 298(2), 195 - 209 Quadruplet codons: implications for code expansion and the specification of translation step size; Moore B et al.; One of the requirements for engineering expansion of the genetic code is a unique codon which is available for specifying the new amino acid . The potential of the quadruplet UAGA in Escherichia coli to specify a single amino acid residue in the presence of a mutant tRNA(Leu) molecule containing the extra nucleotide, U, at position 33.5 of its anticodon loop has been examined . With this mRNA-tRNA combination and at least partial inactivation of release factor 1, the UAGA quadruplet specifies a leucine residue with an efficiency of 13 to 26 % . The decoding properties of tRNA(Leu) with U at position 33.5 of its eight-membered anticodon loop, and a counterpart with A at position 33.5, strongly suggest that in both cases their anticodon loop bases stack in alternative conformations . The identity of the codon immediately 5' of the UAGA quadruplet influences the efficiency of quadruplet translation via the properties of its cognate tRNA . When there is the potential for the anticodon of this tRNA to dissociate from pairing with its codon and to re-pair to mRNA at a nearby 3' closely matched codon, the efficiency of quadruplet translation at UAGA is reduced . Evidence is presented which suggests that when there is a purine base at position 32 of this 5' flanking tRNA, it influences decoding of the UAGA quadruplet . J Mol Biol, 2000 Apr 14, 297(5), 1129 - 43 The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit; Lieberman KR et al.; Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix . We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing . We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA . A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding . Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits . Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains . There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit . J Mol Biol, 2000 Apr 14, 297(5), 1105 - 20 A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin; Schlehuber S et al.; We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules . 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis . From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation . DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a K(D) of 30.2(+/-3.6) nM . The derivative compound digitoxigenin is bound even more tightly, with a K(D) of 2.0(+/-0.52) nM, whereas the steroid glycoside ouabain is not recognized at all . Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant . Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called "anticalins", which provide a promising alternative to recombinant antibody fragments . J Mol Biol, 2000 Apr 14, 297(5), 1037 - 44 A thermodynamic coupling mechanism can explain the GroEL-mediated acceleration of the folding of barstar; Bhutani N et al.; Despite extensive structural and kinetic studies, the mechanism by which the Escherichia coli chaperonin GroEL assists protein folding has remained somewhat elusive . It appears that GroEL might play an active role in facilitating folding, in addition to its role in restricting protein aggregation by secluding folding intermediates . We have investigated the kinetic mechanism of GroEL-mediated refolding of the small protein barstar . GroEL accelerates the observed fast (millisecond) refolding rate, but it does not affect the slow refolding kinetics . A thermodynamic coupling mechanism, in which the concentration of exchange-competent states is increased by the law of mass action, can explain the enhancement of the fast refolding rates . It is not necessary to invoke a catalytic role for GroEL, whereby either the intrinsic refolding rate of a productive folding transition or the unfolding rate of a kinetically trapped off-pathway intermediate is increased by the chaperonin . Am J Respir Crit Care Med, 2000 Apr, 161(4 Pt 1), 1087 - 93 Cardiac contractility is not depressed in early canine endotoxic shock; Pinsky MR et al.; We investigated effects of acute endotoxemia (Escherichia coli endotoxin, 1 mg/kg, intravenously) on left ventricular (LV) function in the first 4 h after induction of endotoxic shock in anesthetized canine preparations (n = 7 each, endotoxin and control groups) . LV pressure and conductance (volume) catheters were used to construct pressure-volume loops . Transient inferior vena cava occlusion was used to rapidly and reversibly alter LV end-diastolic volume . LV contractility was assessed from the slope of the LV end-systolic pressure-volume relationship (Ees) and from preload-recruitable stroke work (PRSW), and from their change (DeltaEes and DeltaPRSW, respectively, measured at 2 and 4 h only), in response to a dobutamine infusion (5 microg/kg/min) . Diastolic function and arterial tone were assessed as the maximal negative change in filling pressure versus time (max -dP/dt), filling rate, and arterial elastance (Ea), respectively . Ees, PRSW, Ea, diastolic function, and hemodynamics were measured hourly . Endotoxemia induced an immediate hypotensive, hyperdynamic, tachycardic state with progressive lactic acidosis . By 2.5 h after endotoxin infusion, heart rate returned to preendotoxin and control levels, but the other changes remained . However, no change occurred in LV Ees, DeltaEes, PRSW, DeltaPRSW, diastolic function, or Ea during the 4-h measurement interval . The cardiovascular collapse seen during the first 4 h of endotoxemia is therefore not due even partly to alterations in LV contractility. Anal Chem, 2000 Apr 1, 72(7), 1611 - 7 Amperometric detection of Escherichia coli heat-labile enterotoxin by redox diacetylenic vesicles on a sol-gel thin-film electrode; Peng T et al.; Supramolecular assemblies (bilayer vesicles) prepared from ferrocenic diacetylene lipid and the cell surface receptor ganglioside GM1 are utilized to construct an amperometric biosensor for Escherichia coli heat-labile enterotoxin on a sol-gel thin-film electrode . The bilayer vesicles adsorbed on the sol-gel film provide an open platform for molecular recognition, while the electrochemical communication between the incorporated redox lipids and the electrode is influenced by the binding of the toxin . Cyclic voltammetric studies suggest a facile redox reaction for the adsorbed supramolecular assembly, which allows the sensor to detect enterotoxin up to 3 ppm (3.6 x 10(-8) M) concentration . The apparent diffusion coefficients for the redox lipids in the assembly were observed to be in the range of 4.73 x 10(-8) -2.30 x 10(-8) cm/s2 . A mechanism of lateral electron transport of redox lipids controlled by biomolecular recognition is proposed. Anal Chem, 2000 Apr 1, 72(7), 1462 - 8 Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry; Zhang CX et al.; A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS) . Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries . Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary . Protein zones are stepwise eluted from the capillary by changing the mobilization conditions . Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary . We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses . The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E . coli lysate proteins using CIEF-ESI-MS . These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS. Clin Lab Haematol, 2000 Feb, 22(1), 49 - 53 Simultaneous occurrence of the 5q- syndrome and multiple myeloma; Rios R et al.; We report a case of the 5q- syndrome with simultaneous occurrence of multiple myeloma, characterized by a very complicated course . To the best of our knowledge, this is the first report of such an association. J Bacteriol, 2000 May, 182(9), 2672 - 4 A common regulator for the operons encoding the enzymes involved in D-galactarate, D-glucarate, and D-glycerate utilization in Escherichia coli; Monterrubio R et al.; Genes for D-galactarate (gar) and D-glucarate (gud) metabolism in Escherichia coli are organized in three transcriptional units: garD, garPLRK, and gudPD . Two observations suggested a common regulator for the three operons . (i) Their expression was triggered by D-galactarate, D-glucarate, and D-glycerate . (ii) Metabolism of the three compounds was impaired by a single Tn5 insertion mapped in the yaeG gene (proposed name, sdaR), outside the D-galactarate and D-glucarate systems . Expression of the sdaR gene is autogenously regulated. J Bacteriol, 2000 May, 182(9), 2619 - 23 Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase; He M et al.; A three-dimensional model of the Streptomyces coelicolor actinorhodin beta-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes . The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site . Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production . (3)H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile. J Bacteriol, 2000 May, 182(9), 2559 - 66 Purification and characterization of the alanine aminotransferase from the hyperthermophilic Archaeon pyrococcus furiosus and its role in alanine production; Ward DE et al.; Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography . The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence . The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor . Optimal activity was found in the pH range of 6 . 5 to 7.8 and at a temperature of over 95 degrees C . The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P . furiosus genome database . The gene was expressed in Escherichia coli, and the recombinant enzyme was purified . The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P . furiosus . The k(cat)/K(m) values for alanine and pyruvate formation were 41 and 33 s(-1) mM(-1), respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine . Northern analysis identified a single 1.2-kb transcript for the aat gene . In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate . The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P . furiosus. J Bacteriol, 2000 May, 182(9), 2498 - 506 Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili; Anantha RP et al.; Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells . A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E . coli . We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA . Here we report the construction and analysis of nonpolar mutations in six genes of the bfp cluster, bfpG, bfpB, bfpC, bfpD, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein . We found that mutations in bfpB, which encodes an outer membrane protein; bfpD, which encodes a putative nucleotide-binding protein; and bfpG and bfpC, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA . The mutation in bfpP, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA . The mutation in bfpH, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA . For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes . We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins . These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly. J Bacteriol, 2000 May, 182(9), 2468 - 75 Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis; Jiang X et al.; RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli . Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end . Here we show that the preference of E . coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region . This property is shared by the related E . coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species . Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E . coli . Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E . coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels. J Bacteriol, 2000 May, 182(9), 2370 - 5 Functional expression in Escherichia coli and membrane topology of porin HopE, a member of a large family of conserved proteins in Helicobacter pylori; Bina J et al.; HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin . In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies . HopE purified from E . coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H . pylori . A model of the membrane topology of HopE was constructed and indicated that this protein formed a beta-barrel with 16 transmembrane amphipathic beta-strands . The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane beta-strands but were tolerated in the adjoining loop regions . Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model . The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane. Int J Biochem Cell Biol, 2000 Apr, 32(4), 405 - 16 Molecular organization, catalytic mechanism and function of serine hydroxymethyltransferase--a potential target for cancer chemotherapy; Rao NA et al.; Serine hydroxymethyltransferase, a pyridoxal-5'-phosphate dependent enzyme, catalyzes the retro-aldol cleavage of serine to yield glycine and the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate to generate 5,10-methylene-H4-folate . The enzyme plays a pivotal role in channeling metabolites between amino acid and nucleotide metabolism . Dihydrofolate reductase and thymidylate synthase have been favorite targets for the development of anticancer drugs . However, developmen