Fold Des, 1998, 3(6), 535 - 48 Functional analysis of the Escherichia coli genome for members of the alpha/beta hydrolase family; Zhang L et al.; BACKGROUND: Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins . Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate . In this work, we have extended a method for active-site identification from predicted protein structures . RESULTS: The structural conservation and variation of the active sites of the alpha/beta hydrolases with known structures were studied . The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions . A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the alpha/beta hydrolase fold family . These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs . 17 ORFs from E . coli were predicted to have hydrolase activity and their putative active-site residues were identified . Most were in agreement with the experiments and results of other database-searching methods . The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the alpha/beta hydrolase family . CONCLUSIONS: The novel feature of our method is that it uses three-dimensional structural information for function prediction . The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms. Biotechnol Appl Biochem, 1999 Feb, 29 ( Pt 1), 31 - 43 Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli; Braun H et al.; The drug delivery system described here is based on the properties of the capsoid or capsid-like structure resulting from the assembly of polyoma virus capsid protein VP1 expressed in Escherichia coli . The capsid protein VP1 was expressed as a fusion protein with a completely removable N-terminal His6 affinity tag . The pentameric morphology of the recombinant VP1 protein was confirmed by electron microscopy after affinity chromatography and factor Xa cleavage under conditions of low ionic strength . The self-assembly of VP1 capsoids can be induced from purified VP1 pentamers by increasing the ionic strength with (NH4)2SO4 . These VP1 capsoid particles were packed in vitro with anti-sense oligonucleotides and plasmid DNA . The loading with DNA was pH-dependent . We observed the highest efficiency at pH 5 . DNase I treatment of particles with encapsidated material showed that 37-55% of the bound oligonucleotides and fragments of 1.5-1.8 kb double-stranded DNA were protected against degradation. J Surg Res, 1999 Jan, 81(1), 81 - 6 Changes in intestinal transit and absorption during endotoxemia are dose dependent; Cullen JJ et al.; BACKGROUND: Septic patients are often intolerant of enteral feedings due to a combination of motility disturbances and impaired absorptive function . Our laboratory has previously demonstrated that endotoxemia results in rapid intestinal transit and decreased jejunal absorption of water, electrolytes, and glucose . We hypothesized that the changes in jejunal transit and absorption during endotoxemia may be dependent on the dose of endotoxin . MATERIALS AND METHODS: Under general anesthesia, rats underwent placement of an internal jugular line, a femoral arterial line, and a 20-cm jejunal Thiry-Vella loop . The jejunal segment was perfused with an isotonic solution containing polyethylene glycol . For 90 min, baseline measurements of blood pressure, heart rate, jejunal absorption of water, electrolytes, and glucose, and jejunal transit were made . Following this baseline period I, rats were given 0.9% NaCl (1 ml/kg) or one of three doses of Escherichia coli lipopolysaccharide (0.5, 1.0, or 5.0 mg/kg) . Studies were then repeated for an additional 90 min . RESULTS: Changes in blood pressure and heart rate were similar among the four groups of animals . Endotoxin decreased water and glucose flux, increased potassium flux, and quickened intestinal transit in a dose-dependent fashion . CONCLUSIONS: We conclude that endotoxemia causes dose-dependent changes in jejunal transit and absorption . The effects of increasing doses of endotoxin on jejunal absorptive and motor function do not appear to be mediated by changes in blood pressure or heart rate . Anal Biochem, 1999 Jan 15, 266(2), 192 - 7 A high-throughput radiometric assay for hepatitis C virus NS3 protease; Cerretani M et al.; A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed . The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein . The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions . To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction . Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin . The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot . Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components . We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way . Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease . Anal Biochem, 1999 Jan 15, 266(2), 167 - 73 Highly efficient green fluorescent protein-based kinase substrates; Yang F et al.; We have developed a general strategy for designing efficient protein substrates of protein kinases by attaching a phosphorylatable peptide sequence to the C-terminus of His6-tagged green fluorescent protein (GFP) . We found that several C-terminal attachment sites in GFP allow for correct presentation of the phosphorylatable tail to a variety of protein kinases . Using this strategy, we have constructed highly efficient GFP-based substrates for Src, c-Abl, protein kinase A, and protein kinase C betaII protein kinases . The engineered GFP substrate for Src (GFP235IYGEFG) is 300 times more efficient than the protein most commonly used as a Src substrate-rabbit muscle enolase . Endocr Res, 1998 Aug-Nov, 24(3-4), 463 - 8 Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis; Arakane F et al.; Steroidogenic acute regulatory protein (StAR) facilitates delivery of cholesterol to the inner mitochondrial membranes . StAR is imported into mitochondria and processed to a mature form by cleavage of the N-terminal mitochondrial targeting sequence . We produced His-tagged (His-tag StAR) constructs lacking the N-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria . His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system . His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells, whereas wild-type StAR was localized to mitochondria . There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes . We established an assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E . coli . Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations . A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein . This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria . Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import. Biochemistry, 1999 Jan 12, 38(2), 848 - 52 Tyrosine 146 of thymidylate synthase assists proton abstraction from the 5-position of 2'-deoxyuridine 5'-monophosphate; Liu Y et al.; Tyr 146 of TS has been proposed to assist in the removal of the proton from the 5-carbon of the pyrimidine in a steady-state intermediate {Hyatt, D . C., Maley, F., and Montfort, W . R . (1997) Biochemistry 36, 4585-4594} . We prepared a replacement set of mutations at position 146 of L . casei TS . The kcat and kcat/Km values of 15 mutants studied were significantly lower than wild-type TS . There was no effect on the Km of dUMP, and only moderate effects on the Km of the cofactor . We concluded that Y146 is not directly involved in substrate binding, but contributes significantly to catalysis . We also examined the Y146 mutants as catalysts for cofactor-independent dehalogenation of BrdUMP, a reaction which simulates early steps of the normal pathway up to and including enzyme-nucleotide covalent adduct formation . Many mutants had activity comparable to the wild-type enzyme, and we concluded that the effects of Tyr 146 mutations occur after the initial covalent adduct is formed . A covalent steady-state intermediate-containing enzyme, dUMP, and cofactor accumulated with Tyr 146 mutants, and could be isolated by SDS-PAGE . The complex was kinetically competent as an intermediate in dTMP formation . Using Y146D and F, it was shown that removal of the C-5 proton from the covalent intermediate was defective . We conclude that in the wild-type enzyme Tyr 146 assists in proton removal from the covalent intermediate . Mutants containing fluorinated tyrosines at position 146 showed an inverse linear correlation of activity versus acidity, again indicating that the basicity of the phenolic oxygen plays an important catalytic role . Speculations of how the poorly basic phenol group might assist proton removal are made in which Tyr 146 acts as a proton conduit to N5 of the cofactor or as a cohort of a water molecule serving as the direct general base catalyst. Biochemistry, 1999 Jan 12, 38(2), 813 - 9 Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli; Sahin-Toth M et al.; Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair {Venkatesan, P., and Kaback, H . R . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 9802-9807} . Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx . In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes . Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx . In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates . Surprisingly, however, no significant exchange or efflux activity is observed . Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax . Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside . Indirect binding assays utilizing substrate protection against {14C}-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG) . The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca . 80 microM) . The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity. Biochemistry, 1999 Jan 12, 38(2), 740 - 50 Magnetic circular dichroism used to examine the interaction of Escherichia coli cytochrome bd with ligands; Borisov V et al.; The interactions of the fully reduced and fully oxidized cytochrome bd from E . coli with ligands CO, NO, and CN- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy . In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b595 and the low-spin heme b558 components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component . The data show that at low concentrations, the ligands bind almost exclusively to heme d . At high concentrations, the ligands begin to interact with the low-spin heme b558 . At the same time, no evidence for significant binding of the ligands to the high-spin heme b595 is revealed in either the reduced or the fully oxidized cytochrome bd complex . The data support the model {Borisov, V . B., Gennis, R . B., and Konstantinov, A . A . (1995) Biochemistry (Moscow) 60, 231-239} according to which the two high-spin hemes d and b595 share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b595. Biochemistry, 1999 Jan 12, 38(2), 696 - 704 Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli; Brautigam CA et al.; The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer . In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen . To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli . Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively . They serve as standards for comparison with other Mn2+- and Zn2+-containing structures . In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J . Mol . Biol . 277, 363-377) . Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution . Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not . It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al . (1997) J . Am . Chem . Soc . 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis. Biochemistry, 1999 Jan 12, 38(2), 667 - 76 Chicken prion tandem repeats form a stable, protease-resistant domain; Marcotte EM et al.; Prion-linked diseases, such as mad cow disease, scrapie, and the human genetic disorder Creutzfeldt-Jakob disease, are fatal neurodegenerative diseases correlated with changes in the secondary structure of neural prion protein . We expressed recombinant chicken prion protein in Escherichia coli and purified the protein to homogeneity . Circular dichroism spectra of the 26 kDa recombinant protein closely resemble those of prion protein purified directly from healthy hamster brain . The chicken prion protein exists as a soluble, monodisperse monomer but can be forced to multimerize following lyophilization and resuspension . We analyzed the chicken prion protein domain structure by proteolysis and show that, unlike the mammalian homologues, the chicken prion protein N-terminal tandem amino acid repeats form a stable, protease-resistant domain . This domain probably represents a physiologically functional unit . As tested by both mass spectrometry and circular dichroism, the mature chicken prion protein does not bind copper, unlike synthetic peptides from the chicken prion N-terminus, suggesting that binding copper is not the physiological activity of the chicken prion . However, copper strongly destabilizes the prion protein and depresses the melting temperature by 30 degreesC, presumably by binding to the unfolded form of the prion protein . The chicken prion N-terminus may have evolved to fold without a cofactor, unlike mammalian prion proteins, whose N-termini are disordered without cofactors such as copper present . Chicken prion offers an alternative to intractable mammalian prions for structural studies of the amino-terminal domain. Biochemistry, 1999 Jan 12, 38(2), 636 - 42 Amino-terminal processing of chemokine ENA-78 regulates biological activity; Nufer O et al.; Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a potent stimulator of neutrophils, inducing a variety of biological responses such as chemotaxis, enzyme release, up-regulation of surface receptors, and intracellular calcium mobilization . Proteolysis of ENA-78 with cathepsin G and chymotrypsin yielded a time-dependent increase in elastase-releasing activity, predicting the formation of truncation products with higher potency than native ENA-78 . To investigate the biological implications of progressive truncation of ENA-78, the N-terminal variants ENA(5-78), ENA(9-78), and ENA(10-78) were cloned and expressed in E . coli . When tested in the neutrophil elastase release assay, the variants ENA(5-78) and ENA(9-78) had a 2-3-fold higher potency than full-length ENA-78, while ENA(10-78) was 3-fold less potent . In the chemotaxis assay, the variant ENA(5-78) exhibited an 8-fold and ENA(9-78) a 2-fold higher potency than native ENA-78 . ENA(10-78), conversely, was 10-fold less potent, but reached a comparable efficacy to ENA-78 at 10(-)7 M concentration . In summary, the rank order in potency with respect to elastase release was ENA(9-78) > ENA(5-78) > ENA-78 > ENA(10-78), while for chemotaxis it was ENA(5-78) > ENA(9-78) > ENA-78 > ENA(10-78) . Variant ENA(5-78) had a higher overall potency and efficiency for chemotaxis than interleukin-8 (IL-8), while ENA(9-78) exhibited a higher efficiency at concentrations of 1-100 nM . The fact that neutrophil cathepsin G produces the stable ENA(9-78) variant in vitro strongly suggests a role for this N-terminal proteolysis during inflammatory processes in vivo. Biochemistry, 1999 Jan 12, 38(2), 589 - 95 Differences in DNA recognition and conformational change activity between boxes A and B in HMG2 protein; Yoshioka K et al.; High mobility group (HMG) 2 is a sequence-nonspecific DNA-binding protein consisting of a repeat of DNA-binding domains called HMG1/2 boxes A and B and an acidic C-terminal . To understand the mode of HMG2 interaction with DNA, we expressed various HMG2 peptides containing HMG1/2 box(es) in Escherichia coli cells and purified them . Gel retardation and DNA supercoiling assay indicated that the region essential for the preferential binding of HMG2 with negatively supercoiled DNA and DNA unwinding activity is located in box B, but not sufficient alone . The flanking C-terminal basic region or box A linked by a linker region is necessary to express activities . The SPR measurements certified that the intrinsic DNA binding affinity of box B is weaker (Kd = 170 microM), and these adjoining regions largely strengthen the affinity (Kd </= 1.2 microM) . In contrast, box A, even in the presence of the adjoining basic linker region, showed no such activities, indicating that boxes A and B are different in their DNA recognition mode . The computer modeling suggested that the side chain of Phe-102 in box B is inserted into the base stack to cause DNA conformational changes, while the side chain of Ala-16 in box A is too small to intercalate . These represent that boxes A and B have similar tertiary structures but their activities for DNA conformational changes obviously differ . Box B is the main region for DNA recognition and conformational changes, and box A must play an assistant to increase its DNA recognition. Biochemistry, 1999 Jan 12, 38(2), 532 - 9 Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4) pyrimidone photodimer by site-directed mutagenesis; Kobayashi H et al.; We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA . In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities . We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli . To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution . We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies . Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region . Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis . Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5 . These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region. Atherosclerosis, 1998 Dec, 141 Suppl 1, S17 - 24 Molecular modelling and the biosynthesis of apolipoprotein B containing lipoproteins; Scott J et al.; APOBEC-1 is the cytidine deaminase . We show by sequence alignment, molecular modelling and mutagenesis, that it is related in crystal structure to the cytidine deaminase of Escherichia coli (ECCDA) . The two enzymes are both homodimers with composite active sites formed with loops from each monomer . In the sequence of APOBEC-1, three gaps compared to ECCDA match the size and contour of the minimal RNA substrate . We propose a model in which the asymmetric binding of one active site to the substrate cytidine which is positioned by the downstream binding of the product uridine and that this helps to target the other active site for deamination. Endocr Res, 1998 Aug-Nov, 24(3-4), 731 - 6 Identification of a novel secretogranin II-derived peptide in the adult and fetal human adrenal gland; Anouar Y et al.; Molecular cloning of secretogranin II (SgII) in different species has revealed the existence of a highly conserved 66-amino acid peptide (EM66) flanked by preserved pairs of basic residues . In the present study we have localized and characterized EM66 in the human adrenal gland . A fusion protein containing the human EM66 peptide was produced in E . coli and used to raise polyclonal antibodies in rabbits . Immunohistochemical staining of human adrenal slices revealed intense labeling of adrenochromaffin cells in the adult and fetal gland . HPLC analysis of adrenal extracts showed the presence of an immunoreactive peak exhibiting the same retention time as recombinant EM66 in both adult and fetus . These data demonstrate that post-translational processing of SgII actually generates a novel peptide in the human adrenal gland . The conservation of the sequence of EM66 in vertebrates and the occurrence of the mature peptide during early ontogenesis of the human adrenal gland strongly suggest that EM66 could exert physiological activities. Endocr Res, 1998 Aug-Nov, 24(3-4), 531 - 9 Structure of adrenodoxin and function in mitochondrial steroid hydroxylation; Bernhardt R et al.; The three-dimensional structure of a truncated mutant of bovine adrenodoxin has been resolved at 1.85 A resolution by MAD . The protein consists of a large core region and a more flexible hairpin loop bearing residues which have been previously described as being involved in redox partner recognition . To study the role of distinct protein domains and amino acids of adrenodoxin in interaction with adrenodoxin reductase (AdR), CYP11A1 and CYP11B1, as well as in electron transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis and produced in Escherichia coli, and their structural and functional properties have been characterized in detail . It could be demonstrated that Tyr82 is located at the edge of the flexible interaction loop of adrenodoxin participating in interactions with AdR and P450s . His56, being close to Tyr82, forms a bridge between the core region of adrenodoxin and the interaction loop . Its role in transmitting changes of the cluster region to the interaction site has also been supported by functional studies . Pro108 of adrenodoxin, the only proline residue contained in the protein and being conserved in this position among several other vertebrate-type ferredoxins, has been demonstrated to be of importance for the correct folding of this protein. Phytochemistry, 1998 Dec, 49(8), 2379 - 82 Alkyl peroxyl radical-scavenging activity of catechins; Nakao M et al.; Alkyl peroxyl radical (ROO.) generated from the reaction between 20 mM t-butyl hydroperoxide (t-BuOOH) and 200 microM hematin could kill E . coli . The minimum concentrations of catechins sufficient to rescue the bacteria treated with ROO . were found to be 70 microM for (-)-epicatechingallate, 100 microM for (-)-epicatechin and 125 microM for (+)-catechin . These values were comparable with the value of alpha-tocopherol, a typical ROO . scavenger . On the other hand, L-ascorbate and beta-carotene revealed about one tenth the scavenging activity of catechins . No scavenging activity was found for superoxide dismutase even at 86 mM . These facts indicate that catechins have high ROO . scavenging activity. Plasmid, 1999 Jan, 41(1), 78 - 81 New cloning vectors with temperature-sensitive replication; Phillips GJ; A series of cloning vectors with conditional, temperature-sensitive replication that are selectable with ampicillin, chloramphenicol, and kanamycin has been constructed . These vectors are derivatives of a pSC101 mutant that can replicate only at low temperatures . The cloning vectors carry a number of unique restriction sites and provide for screening of recombinant plasmids by alpha complementation . These vectors have proven useful for a variety of applications where conditional replication of a recombinant plasmid is desired . Plasmid, 1999 Jan, 41(1), 63 - 9 Instability of pUC19 in Escherichia coli transcription termination factor mutant, rho026; Sozhamannan S et al.; The higher copy number of pUC19, compared to its parent plasmid pBR322, is known to be due to deletion of rop, also known as rom, and to an ori mutation that impedes RNAI:RNAII interaction . pUC19, unlike pBR322, fails to transform E . coli rho mutant rho026 cells . Here we identify two features of pUC19 that contribute to this transformation defect . (1) The pUCori mutation is involved because replacing the pUCori with that of pBR322 restored transformation . (2) Transcription from the lac promoter in pUC19 is important, since deletion or inversion of the promoter or insertion of a transcription terminator (lambdat0) downstream of it restored transformation . Host RNase E activity is responsible for the transformation defect because introduction of an rne-1 allele into rho026 cells suppressed this defect, indicating that RNAI instability due to RNase E is aggravated in the rho026 strain . We suggest that in rho026 cells pUC19 RNAI:RNAII interaction is more impeded than in rho+ cells and Rop/Rom may confer stability by protecting RNAI against RNase E activity because expression of a rom gene inserted into pUC19 restored transformation . Plasmid, 1999 Jan, 41(1), 55 - 62 Allele-specific suppression of ColE1 high-copy-number mutants by a rpoB mutation of Escherichia coli; Yang YL et al.; We have isolated spontaneous rifampicin-resistant mutants from Escherichia coli that showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutants in vivo . The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin . Three host-encoded enzymes, RNase H, DNA polymerase I, and RNA polymerase, are essential to the replication initiation in vitro . To decide whether the activity of RNA polymerase is involved directly in the formation of the persistent hybrid, we screened rifampicin-resistant colonies for suppressors of ColE1 copy-number mutants . Suppressor strain YY572 (rpoB572) changes the 572 residue of the beta subunit of RNA polymerase, encoded by the rpoB gene, from isoleucine to leucine . Another suppressor, YY513 (rpoB513), changes the 513 residue from glutamine to lysine . The other known rifampicin-resistant alleles located at residue 513, rpoB8 and rpoB101, did not show a significant suppression of the copy number of those ColE1 copy-number mutants as rpoB513 . The suppression by rpoB513 on different ColE1 copy-number mutants showed allelic specificity . The possible roles of RNA polymerase in control of ColE1 copy number are discussed . J Mol Biol, 1999 Jan 22, 285(3), 1179 - 94 Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli; Chaudhuri TK et al.; The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk . The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography . Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties . The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one . The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins . The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein . A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region . Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding . The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E . coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect . It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH . J Mol Biol, 1999 Jan 22, 285(3), 1067 - 80 Identification of the acidic residues in the active site of DNA polymerase III; Pritchard AE et al.; The mechanism of nucleotide addition by DNA polymerases involves two metal ions that are coordinated in the active site by conserved acidic residues . The three acidic residues that chelate Mg2+ in the active site of Escherichia coli DNA polymerase III have been identified as Asp401, Asp403, and Asp555 by site-directed mutagenesis . Candidates for mutagenesis were initially chosen based on absolute conservation of acidic residues in an alignment of more than 20 diverse DnaE sequences . Conservative Asp to Glu mutations at positions 401 and 403 reduced the activities of the mutant polymerases 2000 and 333-fold, respectively, from that of the wild-type . The third carboxylate was identified by a series of mutations for each critical candidate . With the exception of Glu, all of the mutations at Asp555 led to severely diminished polymerase activity, while each of the other candidates exhibited several relatively active mutant polymerases . Moreover, only the identified active site mutant polymerases displayed a significant enhancement of activity in Mn2+ compared with Mg2+ . These data suggest a direct involvement of the mutated amino acid in metal ion binding . Anal Biochem, 1999 Jan 1, 266(1), 16 - 22 A dilatancy assay for nucleoid denaturation: the centrifugation-dependent clumping of denatured spermidine nucleoids; Murphy LD et al.; The ability of low centrifugal forces to convert denatured spermidine nucleoids from Escherichia coli into nonsedimentable macroscopic clumps is the basis of a rapid and simple, nonisotopic assay for nucleoid denaturation. Anal Biochem, 1999 Jan 1, 266(1), 9 - 15 BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation; O'callaghan CA et al.; The enzyme BirA is a key reagent because of its ability to biotinylate proteins at a specific residue in a recognition sequence . We report a rapid, efficient, and economical method for the production, purification, and application of this enzyme . The method is easily scaled up and the protein produced is of high purity and can be stored for many months with retention of activity . We have used this enzyme to biotinylate the C termini of membrane proteins, allowing these proteins to be tetramerized by binding to streptavidin . Because of the specificity of the biotinylation at the C terminus, the orientation of the membrane proteins on the streptavidin is equivalent to that of the native protein on the cell surface . These tetrameric proteins can be used to study protein receptor-ligand interactions at the cell surface, and site-specific biotinylation can be used to study proteins in vitro using a defined orientation . Nat Struct Biol, 1999 Jan, 6(1), 80 - 8 Crystal structure of recombinant bovine neurocalcin; Vijay-Kumar S et al.; The crystal structure of calcium-bound unmyristoylated bovine neurocalcin from Escherichia coli has been determined at 2.4 A resolution . The three-dimensional structure reveals a highly compact structure consisting of: (i) two pairs of calcium-binding EF-hands (EF1-EF2 and EF3-EF4); (ii) a calcium ion bound at EF2, EF3 and EF4 sites; and (iii) an EF1-hand that is disabled from calcium-binding due to a Cys-Pro sequence in the Ca2+-binding loop . The crystal structure of neurocalcin resembles photoreceptor recoverin in overall topology, however its EF2- and EF4-hands differ . Recently, neurocalcin in the calcium-bound state has been shown to stimulate mammalian rod outer segment membrane guanylate cyclase . A possible site for cyclase activity based on the three-dimensional structure is discussed. Nat Struct Biol, 1999 Jan, 6(1), 56 - 63 Crystal structure of the outer membrane active transporter FepA from Escherichia coli; Buchanan SK et al.; Integral outer membrane receptors for iron chelates and vitamin B12 carry out specific ligand transport against a concentration gradient . Energy for active transport is obtained from the proton-motive force of the inner membrane through physical interaction with TonB-ExbB-ExbD, an inner membrane complex . Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 A resolution . Two distinct functional domains are revealed: (i) a 22-stranded beta-barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N-terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding . These loops could provide a signaling pathway between the processes of ligand recognition and TonB-mediated transport . The blockage of the pore suggests that the N-terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm. Am J Physiol, 1999 Jan, 276(1 Pt 1), L131 - 6 Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils; Cai GZ et al.; Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to alpha-D-glucosyl residues . It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" region), and a COOH-terminal carbohydrate recognition domain (CRD) . Previous studies demonstrated that SP-D is chemotactic for inflammatory cells . To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose . A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody . The recombinant SP-D CRD was confirmed by amino acid sequencing . Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric . Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10(-10) M equal to the positive control {formyl-Met-Leu-Phe (fMLP) at 10(-8) M} . The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP . The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10(-10) vs . 10(-11) M) . The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD . These preincubations did not affect chemotaxis to fMLP . These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D. J Immunol, 1999 Jan 1, 162(1), 195 - 202 Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants; Tomura T et al.; We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol . Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species . Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli . Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so . It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives . A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not . Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response. Microbiol Immunol, 1998, 42(11), 761 - 71 Studies on the rabies virus RNA polymerase: 2 . Possible relationships between the two forms of the non-catalytic subunit (P protein); Takamatsu F et al.; We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase . The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively . Double labeling experiments with {3H}leucine and {32P}orthophosphate demonstrated that p40 was much more phosphorylated than p37 . Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide . The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion . On the other hand, p40 was apparently detected only in the virion, and little detected in the cells . Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly . We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli . Incubation of 37-kDa products from E . coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin . From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s). Mol Plant Microbe Interact, 1999 Jan, 12(1), 68 - 73 Carbamoylation of azorhizobial Nod factors is mediated by NodU; D'Haeze W et al.; Lipochitooligosaccharides (LCOs) synthesized by Azorhizobium caulinodans ORS571 are substituted at the nonreducing-terminal residue with a 6-O-carbamoyl group . LCO biosynthesis in A . caulinodans is dependent on the nodABCSUIJZnoeC operon . Until now, the role of the nodulation protein NodU in the synthesis of azorhizobial LCOs remained unclear . Based on sequence similarities and structural analysis of LCOs produced by a nodU mutant, a complemented nodU mutant, and Escherichia coli DH5 alpha expressing the nodABCSU genes, NodU was shown to be involved in the carbamoylation step. Appl Biochem Biotechnol, 1998 Aug, 74(2), 95 - 103 Purification of recombinant proteins by chemical removal of the affinity tag; Rais-Beghdadi C et al.; The efficient removal of a N- or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine . Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation . Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification . It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen . The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag . Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins. Am J Hematol, 1999 Jan, 60(1), 6 - 11 Effects of low molecular weight heparin, alone or combined with antithrombin III, on mortality, fibrin deposits and hemostatic parameters in endotoxin-induced disseminated intravascular coagulation in rabbits; Hermida J et al.; The effect of low molecular weight heparin (LMWH) with or without antithrombin III (AT III) has been studied in a rabbit model of disseminated intravascular coagulation (DIC) induced by continuous infusion of 100 microg/kg/hr of Escherichia coli endotoxin for 6 hr . LMWH (5 and 10 IU/kg/hr/6 hr), alone or in combination with AT III (20 U/kg/hr/6 hr), or saline were administered simultaneously with endotoxin . Hemostatic markers at 0, 2, and 6 hr as well as kidney fibrin deposits and the mortality rate at 24 hr were determined . Rabbits receiving only endotoxin showed an impairment in hemostasis, as well as high kidney fibrin deposits and a high mortality rate . LMWH alone did not exert any effect . The simultaneous infusion of LMWH and AT III exerted a beneficial effect on the hemostatic markers and reduced the kidney fibrin deposits as well as the mortality rate in a LMWH dose-dependent manner . Fibrinogen and protein C consumption were significantly higher and renal fibrin deposits more intense in the rabbits that had died in the first 24 hr . There was also a significant positive correlation between kidney fibrin deposits and platelets, fibrinogen, and protein C consumption, taking the whole rabbit population . It is concluded that the simultaneous infusion of LMWH and AT III is useful in this DIC model and would make it possible to reduce significantly the AT III doses used when AT III is given alone. J Bacteriol, 1999 Jan, 181(2), 572 - 6 Effect of ppGpp on Escherichia coli cyclopropane fatty acid synthesis is mediated through the RpoS sigma factor (sigmaS); Eichel J et al.; Strains of Escherichia coli carrying mutations at the relA locus are deficient in cyclopropane fatty acid (CFA) synthesis, a phospholipid modification that occurs as cultures enter stationary phase . RelA protein catalyzes the synthesis of guanosine-3',5'-bisdiphosphate (ppGpp); therefore, ppGpp was a putative direct regulator of CFA synthesis . The nucleotide could act by increasing either the activity or the amount of CFA synthase, the enzyme catalyzing the lipid modification . We report that the effect of RelA on CFA synthesis is indirect . In vitro and in vivo experiments show no direct interaction between ppGpp and CFA synthase activity . The relA effect is due to ppGpp-engendered stimulation of the synthesis of the alternative sigma factor, RpoS, which is required for function of one of the two promoters responsible for expression of CFA synthase. Protein Expr Purif . 1998 Dec;14(3):IV. Papers to appear in forthcoming issues The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site O(B)3 but other KorB-binding sites form destabilizing complexes in the absence of O(B)3. School of Biological Sciences, The University of Birmingham, Edgbaston, UKThe sector of the genome of broad-host-range IncP plasmid RK2 from kb coordinate 54.0 to 60.0 confers an active partitioning phenotype, increasing the segregational stability of low-copy-number unstable plasmids . This Par region encodes the central control operon (korA, incC, korB, korF and korG) and the associated genes kfrA, upf54.8 and upf54.4 . Each ORF in this region was knocked out in turn and it was shown that only incC and korB are needed for the stability phenotype . incC encodes two polypeptides from alternative translational starts . A deletion of the start of the operon showed that only IncC2, the shorter product, is essential for partitioning . Directed mutation or deletion was used to inactivate in turn each of the three KorB-binding sites (O(B)s) which were candidate cis-acting sequences needed for stability . Only inactivation of O(B)3, which lies between upf54.4 and upf54.8, resulted in an increased rate of segregational loss . However, the rate of loss was significantly higher than the rate of loss of the test plasmid carrying none of this RK2 Par region . Either inactivation of korB or deletion of O(B)1 from this O(B)3 mutant resulted in restoration of the loss rate to that expected for the unstable test plasmid alone . Thus KorB can act on O(B)1 to create a complex that either inhibits replication or reduces the effective plasmid copy number, perhaps by promoting pairing between plasmid molecules . This implies that RK2 goes through a cycle of pairing and separation, akin to the mitotic cycle of eukaryotic chromosomes. FEBS Lett, 1998 Dec 18, 441(2), 327 - 30 Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1; Bergman AC et al.; Kinetic properties of the monomeric enzyme dUTPase from herpes simplex virus type 1 (HSV) were investigated and compared to those previously determined for homotrimeric dUTPases of bacterial and retroviral origins . The HSV and Escherichia coli dUTPases are equally potent as catalysts towards the native substrate dUTP with a kcat/K(M) of about 10(7) M(-1) s(-1) and a K(M) of 0.3 microM . However, the viral enzymes are less specific than the bacterial enzyme . The HSV and E . coli dUTPases show the same stereospecificity towards the racemic substrate analogue dUTPalphaS (2'-deoxyuridine 5'-(alpha-thio)triphosphate), suggesting that they have identical reaction mechanisms. FEBS Lett, 1998 Dec 18, 441(2), 247 - 50 Disruption of Escherichia coli transaldolase into catalytically active monomers: evidence against half-of-the-sites mechanism; Schorken U et al.; Disruption of the hydrogen bonding network at the interface of Escherichia coli transaldolase by substitution of R300 to a glutamic acid residue resulted in a monomeric enzyme at basic pH values, with almost no change in the kinetic parameters . The stability of the R300A and R300E mutants towards urea and thermal inactivation is similar to that of the wild-type enzyme . X-ray analysis showed that no structural changes occurred as a consequence of the side chain replacement . This indicates that the quaternary structure is not required for catalytic activity nor does it contribute significantly to the stability of the enzyme . The results are not consistent with a proposed half-of-the-sites reaction mechanism. FEBS Lett, 1998 Dec 18, 441(2), 242 - 6 Analysis of the conserved acidic residues in the regulatory domain of PhoB; Zundel CJ et al.; The PhoB protein from Escherichia coli is a member of the two-component signal transduction pathway that controls an adaptive response to limiting phosphate . Activation involves its phosphorylation on a conserved aspartate . Site-directed mutations were introduced at conserved acidic residues . The E9D, D10E, D10N, E11A, E11D and E11Q mutants were each able to induce alkaline phosphatase under low phosphate growth conditions whereas the E9A, D10A, D53A, D53E and D53N could not . The E9Q mutant was constitutively active . Phosphorylation assays showed that only the E9D, E11A, E11Q and E11D mutants were phosphorylated by acetyl phosphate . Most mutants also displayed defects in magnesium binding. FEBS Lett, 1998 Dec 18, 441(2), 195 - 9 Probing the potential metal binding site in Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (phenylalanine-sensitive); Sundaram AK et al.; The active site residues of the proposed metal binding site of DAH 7-P synthase (phe) were probed by site-directed mutagenesis of C61 to glycine and serine, H64 to glycine, and with the double mutant C61H/H64C . While C61S and C61H/ H64C were inactive, both C61G and H64G were active . All mutants, regardless of enzymatic activity, bound one equivalent of Fe2+ per monomeric unit . Even though C61 and H64 were shown not to be metal ligands for the DAH 7-P synthase (phe), they may provide some of the backbone interactions/secondary structural elements necessary to properly form the metal binding pocket. J Bacteriol, 1999 Jan, 181(2), 681 - 4 Control of Herbaspirillum seropedicae NifA activity by ammonium ions and oxygen; Souza EM et al.; The activity of a truncated form of Herbaspirillum seropedicae NifA in different genetic backgrounds showed that its regulatory domain is involved in nitrogen control but not in O2 sensitivity or Fe dependence . The model for nitrogen control involving PII could thus apply to the proteobacteria at large . NifA may have a role in controlling ADP-ribosylation of nitrogenase in Azospirillum brasilense. J Bacteriol, 1999 Jan, 181(2), 670 - 4 Isolation and characterization of the nikR gene encoding a nickel-responsive regulator in Escherichia coli; De Pina K et al.; Expression of the nickel-specific transport system encoded by the Escherichia coli nikABCDE operon is repressed by a high concentration of nickel . By using random transposon Tn10 insertion, we isolated mutants in which expression of the nik operon became constitutive with respect to nickel . We have identified the corresponding nikR gene which encodes a nickel-responsive regulator . Expression of nikR was partially controlled by Fnr through transcription from the nikA promoter region . In addition, a specific transcription start site for the constitutive expression of nikR was found 51 bp upstream of the nikR gene. J Bacteriol, 1999 Jan, 181(2), 662 - 5 S-methylmethionine metabolism in Escherichia coli; Thanbichler M et al.; Selenium-accumulating Astragalus spp . contain an enzyme which specifically transfers a methyl group from S-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid . Analysis of the amino acid sequence of this enzyme revealed that Escherichia coli possesses a protein (YagD) which shares high sequence similarity with the enzyme . The properties and physiological role of YagD were investigated . YagD is an S-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate . Mutants in yagD which also possess defects in metE and metH are unable to utilize S-methylmethionine for growth, whereas a metE metH double mutant still grows on S-methylmethionine . Upstream of yagD and overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in a metE metH genetic background . Since it displays sequence similarities with amino acid permeases it appears to be the transporter for S-methylmethionine . Methionine but not S-methylmethionine in the medium reduces the amount of yagD protein . This and the existence of four MET box motifs upstream of yfkD indicate that the two genes are members of the methionine regulon . The physiological roles of the ykfD and yagD products appear to reside in the acquisition of S-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis. J Bacteriol, 1999 Jan, 181(2), 563 - 71 The LysR homolog LrhA promotes RpoS degradation by modulating activity of the response regulator sprE; Gibson KE et al.; Synthesis of the OmpF porin of Escherichia coli is regulated in response to environmental and growth phase signals . In order to identify constituents of the various regulatory pathways involved in modulating ompF transcriptional expression, transposon insertion mutagenesis was performed and mutations that increased ompF'-lacZ activity were identified as previously described . Mutations mapping to a previously identified gene of unknown function, lrhA, were obtained . We found that LrhA, a LysR homolog, functions as a regulatory component in the RpoS-dependent growth phase repression of ompF . In addition to altered growth phase regulation of ompF, these lrhA mutants have pleiotropic stationary-phase defects as a result of decreased RpoS levels . We provide evidence that LrhA promotes degradation of RpoS by functioning within a genetic pathway that includes the response regulator SprE and the ClpXP protease . LrhA functions upstream of the other components in the pathway and appears to modulate the activity of SprE. J Bacteriol, 1999 Jan, 181(2), 521 - 30 Septal localization of FtsQ, an essential cell division protein in Escherichia coli; Chen JC et al.; Septation in Escherichia coli requires several gene products . One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain . We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes . The gfp-ftsQ gene complements a null mutation in ftsQ . Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site . Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting . GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains . In addition, the septal localization of ZipA apparently did not require functional FtsQ . Our results indicate that FtsQ is an intermediate recruit to the division site. J Bacteriol, 1999 Jan, 181(2), 477 - 82 Slipped misalignment mechanisms of deletion formation: in vivo susceptibility to nucleases; Bzymek M et al.; Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA . In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE) . Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands . We examined the influence of the 3' exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo . Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3' ends are a common intermediate in both mechanisms of slipped misalignments . Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I . If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms . Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system. J Bacteriol, 1999 Jan, 181(2), 462 - 8 BglF, the Escherichia coli beta-glucoside permease and sensor of the bgl system: domain requirements of the different catalytic activities; Chen Q et al.; The Escherichia coli BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities . In the absence of beta-glucosides, it phosphorylates BglG, a positive regulator of bgl operon transcription, thus inactivating BglG . In the presence of beta-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports beta-glucosides into the cell and phosphorylates them . BglF is composed of two hydrophilic domains, IIAbgl and IIBbgl, and a membrane-bound domain, IICbgl, which are covalently linked in the order IIBCAbgl . Cys-24 in the IIBbgl domain is essential for all the phosphorylation and dephosphorylation activities of BglF . We have investigated the domain requirement of the different functions carried out by BglF . To this end, we cloned the individual BglF domains, as well as the domain pairs IIBCbgl and IICAbgl, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo . We show here that the IIB and IIC domains, linked to each other (IIBCbgl), are required for the sugar-driven reactions, i . e., sugar phosphotransfer and BglG activation by dephosphorylation . In contrast, phosphorylated IIBbgl alone can catalyze BglG inactivation by phosphorylation . Thus, the sugar-induced and noninduced functions have different structural requirements . Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl which occur upon the interaction of BglF with the sugar. J Bacteriol, 1999 Jan, 181(2), 401 - 10 Heat-induced synthesis of sigma32 in Escherichia coli: structural and functional dissection of rpoH mRNA secondary structure; Morita M et al.; The heat shock response in Escherichia coli depends primarily on the increased synthesis and stabilization of otherwise scarce and unstable sigma32 (rpoH gene product), which is required for the transcription of heat shock genes . The heat-induced synthesis of sigma32 occurs at the level of translation, and genetic evidence has suggested the involvement of a secondary structure at the 5' portion (nucleotides -19 to +247) of rpoH mRNA in regulation . We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes . A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo . These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions . The results indicated not only the primary importance of base pairings between the translation initiation region of ca . 20 nucleotides (the AUG initiation codon plus the "downstream box") and the internal region of rpoH mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift. Protein Expr Purif, 1998 Dec, 14(3), 395 - 402 High-level expression of rat farnesyl:protein transferase in Escherichia coli as a translationally coupled heterodimer; Zimmerman KK et al.; Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins . Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the alpha- and beta-subunits are coexpressed, large-scale overexpression of active enzyme has been challenging . We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/L Escherichia coli . Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid . The beta-subunit-coding sequence was placed upstream of the alpha-subunit coding sequence linked by overlapping beta-subunit stop and alpha-subunit start codons . Additionally, the initial 88 codons of the alpha-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E . coli . Since previous attempts at recombinantly expressing FPTase in E . coli from a translationally coupled system have demonstrated that initiation of translation of the alpha-subunit is poor, we propose that the optimization of the codons at the start of the alpha-subunit gene leads to the observed high level of recombinant expression . Protein Expr Purif, 1998 Dec, 14(3), 382 - 6 Green fluorescent protein purification by organic extraction; Yakhnin AV et al.; Green fluorescent protein (GFP) is widely used as an excellent reporter molecule in biochemistry and cell biology . Some biochemical and immunological assays require high-purity GFP . However, the majority of current procedures for GFP purification include multiple time-consuming chromatography steps with a low yield of the desired product or require tag-containing proteins . An alternative method is described for the GFP purification without affinity extensions using organic extraction yielding a highly homogeneous protein indistinguishable in spectroscopic properties from that purified by previous methods . Protein Expr Purif, 1998 Dec, 14(3), 371 - 81 Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli; Banerji S et al.; The CD44 molecule is a widely distributed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan . The ligand-binding site which is located in the membrane distal portion of the molecule encompasses a region of approximately 100 amino acids termed the Link domain, a structural unit that is conserved among members of the Hyaladherin superfamily which includes cartilage link protein, aggrecan, and tumor necrosis factor-stimulated gene-6 (TSG-6) . In contrast to these other Hyaladherins, however, the ligand-binding domain of CD44 appears to extend beyond the Link domain to involve additional basic residues located toward the membrane proximal region . Furthermore, recent molecular modeling studies indicate that within the CD44 Link domain itself, the spatial arrangement of critical residues involved in HA binding is likely to differ significantly from the prototypic TSG-6 Link module . In order to obtain material to solve the CD44 solution structure we have developed an optimized method for the expression and purification of functionally active CD44 ectodomains encompassing both the Link module and the additional downstream HA-binding residues in Escherichia coli . Here we describe the details of the method which involves solubilization of recombinant CD44 from inclusion bodies in 8 M urea, followed by refolding and purification of intact monomers using size-exclusion and reverse-phase chromatography . We show the method yields CD44 molecules that (1) retain reactivity with a panel of conformation-sensitive antibodies, (2) possess similar hyaluronan-binding characteristics to authentically folded CD44 molecules expressed in eukaryotic cells, and (3) display one-dimensional NMR spectra that indicate the presence of a single conformational species . This method should enable sufficient amounts of functional CD44 Link module to be produced for comprehensive structural analyses by multidimensional NMR spectroscopy . Protein Expr Purif, 1998 Dec, 14(3), 343 - 52 Recombinant human cytomegalovirus protease with a C-terminal (His)6 extension: purification, autocatalytic release of the mature enzyme, and biochemical characterization; Tomasselli AG et al.; Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics . To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli . The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site . The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence . In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation . The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E . coli as inclusion bodies . After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography . The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein . This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus . Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy . The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E . coli . In addition, the refolded CMV PR could be crystallized for X-ray diffraction . Protein Expr Purif, 1998 Dec, 14(3), 335 - 42 Production of leptin in Escherichia coli: a comparison of methods; Varnerin JP et al.; A procedure is described for gram-scale refolding of Escherichia coli-derived human leptin inclusion bodies . Refolding was achieved by gradually reducing denaturant using a diafiltration method . Refolded leptin is characterized by in vivo modulation of food intake, reduction in body weight, and lowering of insulin and glucose levels in ob/ob mice . In addition, refolded leptin is characterized by radioimmunoassay (RIA) and activation of the leptin receptor in a cell-based assay . For comparison we also refolded leptin by a simple dilution method and produced periplasmic derived leptin, which did not require ex vivo folding . Leptin produced by these three methods and leptin obtained from commercial sources were compared using the RIA and the cell-based assay and appeared to be of comparable quality and potency . Arch Biochem Biophys, 1999 Jan 15, 361(2), 302 - 8 Amino acid substitutions in the a subunit affect the epsilon subunit of F1F0 ATP synthase from Escherichia coli; Gardner JL et al.; Amino acid substitutions at many positions in the a subunit of F1F0 ATP synthase result in impaired proton translocation and altered catalytic activity . In this work, we demonstrate that amino acid substitutions in the a subunit affect the epsilon subunit . In mutant F1F0 ATP synthases, the epsilon subunit was studied by determining its sensitivity to proteolysis and by chemical crosslinking under conditions of active turnover and in quiescent enzyme . Like native F1F0 ATP synthase, the epsilon subunit in enzymes carrying either the aarg-210-->ile or agly-218-->asp substitutions proved resistant to trypsin digestion during ATP hydrolysis . In each case, the epsilon subunit was rapidly digested in the presence of a nonhydrolyzable ligand, but this did not result in the activation of hydrolytic activity typically seen in wild-type enzyme . In enzyme carrying the aala-217-->arg substitution, the trypsin digestion of the epsilon subunit occurred regardless of ligand and was accompanied by a limited hydrolytic activation . Relative to the native F1F0 ATP synthase, the aala-217-->arg substitution resulted in reduced efficiency of crosslinking between the epsilon and beta subunits using 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide . These observations indicate that the structural changes resulting from amino acid substitutions in the a subunit are propagated to the epsilon subunit and are specific to the individual substitutions . Arch Biochem Biophys, 1999 Jan 15, 361(2), 195 - 201 Identification and cloning of rat galectin-2: expression is predominantly in epithelial cells of the stomach; Oka T et al.; A complementary DNA clone preferentially expressed in the gastrointestinal tract was obtained from a rat stomach library . The protein coded by the clone had a single carbohydrate recognition domain having conserved motifs for beta-galactoside binding and showed 67% amino acid identity with human galectin-2 . The recombinant protein synthesized in Escherichia coli could bind to an asialofetuin column and was eluted with beta-galactopyranoside . From these observations, we named the protein rat galectin-2 coded by the cDNA . The rat galectin-2 was predominantly expressed in the epithelial cells of stomach . Thus this protein may form a mucin layer cross-linking with the beta-galactoside moiety of glycoproteins . Arch Biochem Biophys, 1999 Jan 1, 361(1), 106 - 12 Cloning, overproduction, and purification of native and mutant recombinant yeast orotate phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetime; Witte JF et al.; The gene for orotate phosphoribosyltransferase from Saccharomyces cerevisiae has been subcloned into an Escherichia coli overexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step . We were able to repeat the published (J . Victor, L . B . Greenberg, and D . L . Sloan J . Biol . Chem . 254, 2647-2655, 1979) . 32PPi/5-phosphorylribose 1-alpha-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well . However, when contaminating orotidine 5'-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished . Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments . Arch Biochem Biophys, 1999 Jan 1, 361(1), 94 - 100 Six putative IQ motifs of the recombinant chicken intestinal brush border myosin I are involved in calmodulin binding; Khoroshev MI et al.; Chicken brush border myosin I has up to six IQ sequence motifs at which it may bind calmodulin . To determine the relative contributions of these motifs to calmodulin binding, fusion deletion fragments were expressed in Escherichia coli and their ability to bind calmodulin was assessed by the gel overlay technique . The first three N-terminal IQ sites showed strong binding with calmodulin . Surprisingly, the last three incomplete IQ motifs also contributed substantial calmodulin binding . The first and fourth IQ sites bound calmodulin but tended to reduce binding in combination with other sites . The data indicate that interactions among all six IQ motifs contribute to the ability of myosin I to bind calmodulin . Anal Biochem, 1998 Dec 15, 265(2), 299 - 307 Purine nucleoside phosphorylase: its use in a spectroscopic assay for inorganic phosphate and for removing inorganic phosphate with the aid of phosphodeoxyribomutase; Nixon AE et al.; The kinetics of the phosphorolysis of 7-methylated guanosine analogues catalyzed by purine nucleoside phosphorylase has been analyzed to understand the use of this system as a "Pi mop" to remove Pi from solutions and as a spectroscopic assay for Pi at micromolar concentrations . An expression system was developed for the phosphorylase from Escherichia coli: this protein (subunit molecular mass 26 kDa) and one from a commercial source (29 kDa) were used in this study . Rates of >50 s-1 were obtained for the phosphorolysis at 30 degrees C, so that when the phosphorylase is coupled to the phosphatase being studied, rates of Pi release from the phosphatase can be measured close to this rate . The kinetic mechanism appears to obey the Michaelis-Menten model in the steady state with the bond cleavage rate limiting . Slow hydrolysis of ribose-1-phosphate to Pi catalyzed by the phosphorylase limits the efficiency of the Pi mop . To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the Pi mop to remove large amounts of Pi quantitatively . Acyclovir diphosphate provides a simple method to switch off the Pi mop as it is a tight inhibitor (Kd 12 nM) of purine nucleoside phosphorylase . Environ Mol Mutagen, 1998, 32(4), 325 - 30 Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay; Zimmer DM et al.; We examined several experimental parameters of the lambda cI/cII transgenic mutation assay . In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice . Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer . Storage of packaged phage for 28 days at 4 degrees C did not affect titer . The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested . Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E . coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C . The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation. J Biol Chem, 1999 Jan 15, 274(3), 1449 - 57 Molecular recognition in a post-translational modification of exceptional specificity . Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase; Chapman-Smith A et al.; We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase . We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro . This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins . Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants . The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly . Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance . Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins. J Virol, 1999 Feb, 73(2), 1649 - 54 Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli; Ferrari E et al.; Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents . In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents . Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B . Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others . For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity . Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner . Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases. J Virol, 1999 Feb, 73(2), 1609 - 16 A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement; Padidam M et al.; Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles . Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants . In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus . To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5) . The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA . The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA . The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication . ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms . In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei . We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection. J Virol, 1999 Feb, 73(2), 1399 - 410 Mutational analysis of the avian adenovirus CELO, which provides a basis for gene delivery vectors; Michou AI et al.; The avian adenovirus CELO is being developed as a gene transfer tool . Using homologous recombination in Escherichia coli, the CELO genome was screened for regions that could be deleted and would tolerate the insertion of a marker gene (luciferase or enhanced green fluorescent protein) . For each mutant genome, the production of viable virus able to deliver the transgene to target cells was monitored . A series of mutants in the genome identified a set of open reading frames that could be deleted but which must be supplied in trans for virus replication . A region of the genome which is dispensable for viral replication and allows the insertion of an expression cassette was identified and a vector based on this mutation was evaluated as a gene delivery reagent . Transduction of avian cells occurs at 10- to 100-fold greater efficiency (per virus particle) than with an adenovirus type 5 (Ad5)-based vector carrying the same expression cassette . Most important for gene transfer applications, the CELO vector transduced mammalian cells as efficiently as an Ad5 vector . The CELO vector is exceptionally stable, can be grown inexpensively in chicken embryos, and provides a useful alternative to Ad5-based vectors. J Virol, 1999 Feb, 73(2), 1392 - 8 Coxsackievirus and adenovirus receptor amino-terminal immunoglobulin V-related domain binds adenovirus type 2 and fiber knob from adenovirus type 12; Freimuth P et al.; The extracellular region of the coxsackievirus and adenovirus receptor (CAR) is predicted to consist of two immunoglobulin (Ig)-related structural domains . We expressed the isolated CAR amino-terminal domain (D1) and a CAR fragment containing both extracellular Ig domains (D1/D2) in Escherichia coli . Both D1 and D1/D2 formed complexes in vitro with the recombinant knob domain of adenovirus type 12 (Ad12) fiber, and D1 inhibited adenovirus type 2 (Ad2) infection of HeLa cells . These results indicate that the adenovirus-binding activity of CAR is localized in the amino-terminal IgV-related domain and confirm our earlier observation that Ad2 and Ad12 bind to the same cellular receptor . Preliminary crystallization studies suggest that complexes of Ad12 knob bound to D1 will be suitable for structure determination. J Virol, 1999 Feb, 73(2), 1046 - 53 A replication-incompetent adenovirus vector with the preterminal protein gene deleted efficiently transduces mouse ears; Moorhead JW et al.; Adenoviruses offer great potential as gene therapy agents but are limited by the strong inflammatory response that occurs in response to the recombinant virus . Since the degree of inflammation correlates in part with the potential of the viral vector for replication, we constructed a preterminal protein (pTP) deletion mutant adenovirus type 5 vector, Ad5dl308DeltapTPbeta-gal, that is replication incompetent due to deletion of the pTP gene and that has the E1 genes replaced by the Escherichia coli lacZ reporter gene under the control of the cytomegalovirus major immediate-early promoter . This virus was compared with a first-generation, replication-defective adenovirus vector, Ad5dl308beta-gal, that is isogenic except that it contains a wild-type pTP gene . To examine transduction efficiency and induction of inflammation, we developed a novel system involving intradermal injection of BALB/c mouse ears . Mouse ears can be accurately measured to determine the degree of edema as an indirect measurement of inflammation . Edema and inflammation were induced in a dose- and time-dependent manner by both viruses and correlated well . LacZ activity correlated inversely with edema and inflammation . The pTP-defective vector Ad5dl308DeltapTPbeta-gal transduced mouse ears much more efficiently and induced edema and inflammatory cell infiltration approximately 10-fold less efficiently than the first-generation vector Ad5dl308beta-gal . The diminished inflammatory response and increased efficiency of transduction observed with Ad5dl308DeltapTPbeta-gal indicate its promise as a gene therapy agent for other tissues . The results also demonstrate that the mouse ear model offers potential for the study of adenovirus-induced inflammation because of the ready access of the ears, the relative ease of continuous measurement, and the sensitivity to adenovirus transducing vectors. Lab Invest, 1998 Dec, 78(12), 1615 - 23 S19 ribosomal protein cross-linked dimer causes monocyte-predominant infiltration by means of molecular mimicry to complement C5a; Nishiura H et al.; S19 ribosomal protein is a component of the protein-producing machinery, ribosome . When recombinant S19 proteins were cross-linked intermolecularly by plasma transglutaminase (coagulation factor XIIIa), this homodimer newly exhibited monocyte chemotactic activity . This effect was specific to monocytes . The S19 protein homodimer shared the immunoreactivity with the complement-derived chemotactic factor, component 5a (C5a) . Monocytes pretreated with an anti-C5a receptor antibody or with a synthetic C5a receptor antagonist responded poorly in chemotaxis to this homodimer . These data indicate that the S19 protein homodimer possesses a 3-dimensional structure similar to C5a in terms of the immunologic epitope and the receptor ligand, although homology between their primary structures is only 4% . In contrast, the S19 protein homodimer did not attract polymorphonuclear leukocytes . In addition, the homodimer inhibited a chemotactic response of polymorphonuclear leukocytes to C5a in vitro and in vivo as a receptor antagonist . Furthermore, the S19 protein homodimer competitively inhibited the binding of radiolabeled C5a to polymorphonuclear leukocytes . The S19 protein homodimer with these opposite effects in the leukocyte chemotaxis seems to induce the monocyte/macrophage predominant infiltration in chronic inflammation. Glycoconj J, 1998 Jun, 15(6), 605 - 13 Inhibition of the type 1 fimbriae-mediated adhesion of Escherichia coli to erythrocytes by multiantennary alpha-mannosyl clusters: the effect of multivalency; Lindhorst TK et al.; Alpha-mannosyl glycoclusters and glycodendrimers were tested as multivalent inhibitors of the type 1 (mannose-specific) fimbriae of a recombinant E . coli HB101 strain . Inhibition of haemagglutination of guinea pig erythrocytes was determined on microtiter plates . The effect of multivalency is pronounced for up to three mannosyl residues in the molecule, whereas larger derivatives do not have an appreciable effect on binding to the fimbrial carbohydrate binding domain . The best glycoclusters tested reach the binding potency of the known potent inhibitor pNPMan (3) . The results support the idea of a monovalent recognition site at the adhesive protein FimH, which might best accommodate molecules with the size of a trisaccharide or those which expose up to three alpha-mannosyl residues, such as the glycocluster 8 . The results obtained with the thiourea-bridged alpha-mannosyl clusters, possessing defined sugar valencies, facilitate the development of high affinity inhibitors of the fimbrial lectin on type 1 fimbriae. Biol Pharm Bull, 1998 Dec, 21(12), 1282 - 5 Biochemical characterization of recombinant HIV-1 reverse transcriptase (rRT) as a glycyrrhizin-binding protein and the CK-II-mediated stimulation of rRT activity potently inhibited by glycyrrhetinic acid derivative; Harada S et al.; By means of successive Mono Q and glycyrrhizin (GL)-affinity column chromatography (HPLC), recombinant HIV-1 RT (rRT) was purified to apparent homogeneity from the Superdex 200 pg fraction of the crude protein extract of E . coli BL21 transfected with pET 21a(+)/HIV-1 PR-RT . It was found that (i) rRT functioned as an effective phosphate acceptor for recombinant human casein kinase II (rhCK-II) in vitro; (ii) this phosphorylation was inhibited by anti-HIV-1 substances {a glycyrrhetinic acid derivative (oGA) and quercetin} and a high dose (100 microM) of GL; (iii) RNA-dependent DNA polymerase (RDDP) activity was stimulated about 2.5-fold after full phosphorylation of rRT by rhCK-II; and (iv) oGA as well as NCS-chromophore effectively prevented the CK-II-mediated stimulation of RDDP activity . These results suggest that the anti-HIV-1 effect of oGA may be involved in the selective inhibition of the CK-II-mediated stimulation of HIV-1 RT at the cellular level. Plant J, 1998 Nov, 16(4), 515 - 22 An oligo selection procedure for identification of sequence-specific DNA-binding activities associated with the plant defence response; Wang Z et al.; Sequence-specific DNA-binding (SSDB) factors play central roles in transcription, DNA replication, recombination and repair . This report describes a simple procedure for high-throughput identification of SSDB activities without prior knowledge of their target genes or binding sequences . The procedure starts with a population of completely random oligo(nucleotide) sequences and selects for those oligo molecules that specifically bind to cellular SSDB factors by use of common gel-retardation assays and PCR . Amplification and subsequent cloning of these selected oligo molecules result in the establishment of oligo DNA libraries enriched in DNA molecules containing specific sequences recognized by SSDB factors . These oligo libraries can be rapidly screened to identify a large number of SSDB activities, including those that are differentially regulated by developmental and environmental signals . With identified oligo DNA as probes, the corresponding SSDB factors can be isolated and analysed with respect to their structures, regulation and functions . Using this procedure, we have identified approximately 100 SSDB activities from tobacco leaves, including seven that are differentially regulated during the tobacco mosaic virus-induced hypersensitive response in resistant tobacco plants. Plant J, 1998 Nov, 16(4), 421 - 31 cDNA cloning, gene expression and subcellular localization of anthocyanin 5-aromatic acyltransferase from Gentiana triflora; Fujiwara H et al.; Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less under-stood modification reactions during anthocyanin biosynthesis . Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins . A full-length cDNA encoding the anthocyanin 5-aromatic acyltransferase (5AT) (EC 2.3.1.153) that acylates the glucose bound at the 5-position of anthocyanidin 3,5-diglucoside was isolated from petals of Gentiana triflora on the basis of the amino acid sequence of the purified enzyme . The isolated full-length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52,736 . The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA-mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multi-functional acyltransferases (St-Pierre et al . (1998) Plant J . 14, 703-713) . The cDNA was expressed in Escherichia coli and yeast, and confirmed to encode 5AT . The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT . Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of G . triflora . RNA blot analysis showed that the 5AT gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes . Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol. Plant J, 1998 Nov, 16(3), 335 - 43 A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine; Guillen P et al.; Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines . It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity . We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA . This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol . The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii . Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates . However, the highest affinity was observed with 3-substituted benzaldehydes . Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin . This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback. J Biochem (Tokyo), 1999 Jan, 125(1), 186 - 93 An azide-insensitive superoxide dismutase from a hyperthermophilic archaeon, Sulfolobus solfataricus; Yamano S et al.; The superoxide dismutase (SOD) gene of Sulfolobus solfataricus, a hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized . When the protein was expressed in E . coli, it formed a homodimer that contained both Mn and Fe . Metal reconstitution experiments of the SOD with Fe or Mn showed that only the Fe-reconstituted SOD was active . Substitution of Tyr88 to Phe did not affect the metal specificity of the enzyme . The Fe-reconstituted SOD was extremely resistant to thermal denaturation; e.g . 96% of the initial activity was retained after heating at 95 degreesC for 2 h . Fe-reconstituted SOD was not inhibited by azide, but fluoride inhibition was observed . This suggests that some steric hindrance in the substrate funnel of the enzyme prevents the access of N3- but allows O2- and F- access to the active site. J Biochem (Tokyo), 1999 Jan, 125(1), 166 - 72 1,5-Anhydroglucitol promotes glycogenolysis in Escherichia coli; Shiga Y et al.; Glycogen is a storage compound that provides both carbon and energy, but the mechanism for the regulation of its metabolism has not been fully clarified . Recently, we found a new glycogenolytic pathway in rat liver in which glycogen is first metabolized to 1, 5-anhydrofructose (AnFru) and then to 1,5-anhydroglucitol (AnGlc-ol) . Because the amounts of glycogen and AnFru are closely related in various rat organs and the second reaction, AnFru to AnGlc-ol, is strongly inhibited in the presence of glucose, we expected that this pathway might play a regulatory role in glycogen metabolism . Here we evaluate the expected involvement of AnGlc-ol and AnFru in the regulatory mechanism in Escherichia coli C600 . Having established the presence of this new glycogenolytic pathway in E . coli C600, we further show that the conversion of AnFru to AnGlc-ol is activated only after the exhaustion of glucose in the medium, and that as little as 5 microM AnGlc-ol in the medium acutely accelerates the degradation of glycogen by 40% . We consider the role of AnGlc-ol in the mechanism that controls glycogen metabolism in E . coli to be as follows . When glucose is abundant, E . coli accumulate glycogen, a fraction of which is steadily degraded so that the amount of AnFru is about 1/1,000 of glycogen on a weight basis . When glucose is depleted and the demand for glycogen utilization is elevated, AnFru, which has accumulated mostly in the medium, is promptly taken up and converted to AnGlc-ol, which accelerates glycogen degradation . We also suggest the possibility that AnGlc-ol is one of the extracellular signaling molecules for bacteria. J Biochem (Tokyo), 1999 Jan, 125(1), 138 - 42 Characterization of the ubiquinol oxidation sites in cytochromes bo and bd from Escherichia coli using aurachin C analogues; Miyoshi H et al.; Natural aurachin C is the most potent inhibitor of oxidation of ubiquinols by cytochromes bo and bd from Escherichia coli . To probe the structural properties of the substrate oxidation site in the ubiquinol oxidases, we synthesized a systematic set of aurachin C analogues (N-hydroxy-4-quinolone derivatives) and examined how their structure affects their activity towards cytochromes bo and bd, which are structurally unrelated . We found that the presence of the 3-methyl group of the 2-n-decyl and 2-n-undecyl derivatives increased the inhibitory potency towards both enzymes, probably due to a local steric congestion that allows favorable interaction of the alkyl tail with the enzyme . Increase in the chain length of the 3-alkyl tail of the 2-n-undecyl derivatives decreased the inhibitory potency only in cytochrome bo, indicating that the binding site for the alkyl tails of cytochrome bo is smaller than that of cytochrome bd . Based on these findings, we discuss the differences in the molecular mechanism of substrate oxidation by these two terminal ubiquinol oxidases. J Biochem (Tokyo), 1999 Jan, 125(1), 24 - 6 Crystallization and preliminary X-ray diffraction studies of a replication initiator protein (RepE54) of the mini-F plasmid complexed with iteron DNA; Komori H et al.; A replication initiator protein (RepE54) complexed with iteron DNA at its binding site was crystallized by the hanging drop vapor diffusion method . The crystals belong to monoclinic space group C2 with unit cell dimensions of a = 108.4 A, b = 81.9 A, c = 73.9 A, and beta = 121.5 degrees, where one molecule of the protein-DNA complex exists per asymmetric unit . They diffract X-rays up to 2.6 A resolution with synchrotron radiation. J Neurosci, 1999 Jan 15, 19(2), 716 - 25 Lipopolysaccharide injected into the cerebral ventricle evokes fever through induction of cyclooxygenase-2 in brain endothelial cells; Cao C et al.; Activation of the arachidonic acid cascade is an essential step for the development of fever during brain inflammation . We investigated the brain sites where this activation takes place by use of a rat model of brain inflammation . Intracerebroventricular administration of lipopolysaccharide but not of its vehicle evoked fever . The fever was markedly suppressed when the rats had been pretreated with a cyclooxygenase-2-specific inhibitor . In situ hybridization and immunohistochemical studies revealed that cyclooxygenase-2 mRNA and its protein were induced by lipopolysaccharide in blood vessels near the cerebral ventricles and in those in the subarachnoidal space . Double immunohistochemical staining revealed that these cyclooxygenase-2-positive cells were mostly endothelial cells . The time course of fever and that of cyclooxygenase-2 induction in the endothelial cells were in parallel . Cyclooxygenase-2 mRNA in a certain type of telencephalic neurons was also upregulated by the intracerebroventricular administration, but this neuronal response occurred both in vehicle-injected rats and in lipopolysaccharide-injected ones to the same extent . Therefore, the neuronal response was not essential to the development of fever . These results suggest that brain endothelial cells play a crucial role in the development of fever during brain inflammation by activating their arachidonic acid cascade. J Biol Chem, 1999 Jan 15, 274(3), 1828 - 34 Isolation of a chinese hamster ovary (CHO) cDNA encoding phosphatidylglycerophosphate (PGP) synthase, expression of which corrects the mitochondrial abnormalities of a PGP synthase-defective mutant of CHO-K1 cells; Kawasaki K et al.; Phosphatidylglycerophosphate (PGP) synthase catalyzes the first step in the cardiolipin (CL) branch of phospholipid biosynthesis in mammalian cells . In this study, we isolated a Chinese hamster ovary (CHO) cDNA encoding a putative protein similar in sequence to the yeast PGS1 gene product, PGP synthase . The gene for the isolated CHO cDNA was named PGS1 . Expression of the CHO PGS1 cDNA in CHO-K1 cells and production of a recombinant CHO PGS1 protein with a N-terminal extension in Escherichia coli resulted in 15-fold and 90-fold increases of PGP synthase specific activity, respectively, establishing that CHO PGS1 encodes PGP synthase . A PGP synthase-defective CHO mutant, PGS-S, isolated previously (Ohtsuka, T., Nishijima, M., and Akamatsu, Y . (1993) J . Biol . Chem . 268, 22908-22913) exhibits striking reductions in biosynthetic rate and cellular content of phosphatidylglycerol (PG) and CL and shows mitochondrial morphological and functional abnormalities . The CHO PGS-S mutant transfected with the CHO PGS1 cDNA exhibited 620-fold and 7-fold higher PGP synthase activity than mutant PGS-S and wild type CHO-K1 cells, respectively, and had a normal cellular content and rate of biosynthesis of PG and CL . In contrast to mutant PGS-S, the transfectant had morphologically normal mitochondria . When the transfectant and mutant PGS-S cells were cultivated in a glucose-depleted medium, in which cellular energy production mainly depends on mitochondrial function, the transformant but not mutant PGS-S was capable of growth . These results demonstrated that the morphological and functional defects displayed by the PGS-S mutant are due directly to the reduced ability to make normal levels of PG and/or CL. J Biol Chem, 1999 Jan 15, 274(3), 1745 - 52 Cross-induction of glc and ace operons of Escherichia coli attributable to pathway intersection . Characterization of the glc promoter; Pellicer MT et al.; The metabolic pathways specified by the glc and ace operons in Escherichia coli yield glyoxylate as a common intermediate, which is acted on by two malate synthase isoenzymes: one encoded by glcB and the other by aceB . Null mutations in either gene exhibit no phenotype, because of cross-induction of the ace operon by glycolate and the glc operon by acetate . In this study, the regulation of the glc operon, comprising the structural genes glcDEFGB, was analyzed at the molecular level . This operon, activated by growth on glycolate, is transcribed as a single message and is under the positive control of GlcC encoded by a divergent gene . Expression of the glc operon is strongly dependent on the integration host factor (IHF) and is repressed by the global respiratory regulator ArcA-P . In vitro gel-shift experiments demonstrated direct binding of the promoter DNA to IHF and ArcA-P . Mutant analysis indicated that cross-induction of the glc operon by acetate is mediated by the GlcC protein that recognizes the compound as an alternative effector . The similar pattern of regulation of the Glc and Ace systems by IHF and ArcA-P ensures their effective cross-induction. J Biol Chem, 1999 Jan 15, 274(3), 1657 - 66 Molecular parameters of type IV alpha-internexin and type IV-type III alpha-internexin-vimentin copolymer intermediate filaments; Steinert PM et al.; During neuronal development, a dynamic replacement mechanism occurs in which the type VI nestin and type III vimentin intermediate filament proteins are replaced by a series of type IV proteins beginning with alpha-internexin . We have explored molecular details of how the type III to type IV replacement process may occur . First, we have demonstrated by cross-linking experiments that bacterially expressed forms of alpha-internexin and vimentin form heterodimer molecules in vitro that assemble into copolymer intermediate filaments . We show using a urea disassembly assay that alpha-internexin molecules are likely to be more stable than those of vimentin . Second, by analyses of the induced cross-links, we have determined the axial lengths of alpha-internexin homodimer and alpha-internexin-vimentin heterodimer molecules and their modes of alignments in filaments . We report that these dimensions are the same as those reported earlier for vimentin homopolymer molecules and, by implication, are also the same for the other neuronal type IV proteins . These data suggest that during neuronal development, alpha-internexin molecules are readily assimilated onto the pre-existing vimentin cytoskeletal intermediate filament network because the axial lengths and axial alignments of their molecules are the same . Furthermore, the dynamic replacement process may be driven by a positive equilibrium due to the increased stability of the alpha-internexin network. J Biol Chem, 1999 Jan 15, 274(3), 1342 - 8 Characterization and binding specificity of the monomeric STAT3-SH2 domain; Haan S et al.; Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction . STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains . The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus . Here we describe the expression, purification, and refolding of the STAT3-SH2 domain . Proper folding of the isolated protein was proven by circular dichroism and fluorescence spectroscopy . The STAT3-SH2 domain undergoes a conformational change upon dimerization . Using an enzyme-linked immunosorbent assay we demonstrate that the monomeric domain binds to specific phosphotyrosine peptides . The specificity of binding to phosphotyrosine peptides was assayed with the tyrosine motif encompassing Tyr705 of STAT3 and with all tyrosine motifs present in the cytoplasmic tail of the signal transducer gp130. J Biol Chem, 1999 Jan 15, 274(3), 1320 - 5 Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an enzyme not found in nature; Mouratou B et al.; Tyrosine phenol-lyase (TPL), which catalyzes the beta-elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the alpha-family of vitamin B6-dependent enzymes . To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL . Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs . Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme . The double mutation R100T/V283R of TPL increased the beta-elimination activity toward dicarboxylic amino acids at least 10(4)-fold . Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g . formate in the case of L-aspartate . The activity toward L-aspartate (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine . beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) were the only reactions observed . Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity . Dicarboxylic amino acid beta-lyase is an enzyme not found in nature. J Biol Chem, 1999 Jan 15, 274(3), 1306 - 12 The Escherichia coli MutL protein physically interacts with MutH and stimulates the MutH-associated endonuclease activity; Hall MC et al.; All possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins . A two-hybrid screen previously identified a physical interaction between MutL and UvrD . Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected . A series of truncations from the NH2 and COOH termini of MutL demonstrated that the COOH-terminal 218 amino acids were sufficient for the two-hybrid interaction with MutH . Removal of a small number of residues from either the NH2 or COOH termini of MutH eliminated the two-hybrid interaction with MutL . Protein affinity chromatography experiments confirmed that MutL, but not MutS, physically associates with MutH . Furthermore, MutL greatly stimulated the d(GATC)-specific endonuclease activity of MutH in the absence of MutS and a mispaired base . Stimulation of the MutH-associated endonuclease activity by MutL was dependent on ATP binding but not ATP hydrolysis . Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP . These results suggest that MutL activates the MutH-associated endonuclease activity through a physical interaction during methyl-directed mismatch repair in Escherichia coli. J Biol Chem, 1999 Jan 15, 274(3), 1248 -