Protein Expr Purif, 2001 Nov, 23(2), 296 - 300 High-level expression and single-step purification of human tryptophanyl-tRNA synthetase; Xu F et al.; Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL . The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography . The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction . The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture . The His(6) tag at C terminus had little effect on the binding ability of its substrates . Protein Expr Purif, 2001 Nov, 23(2), 289 - 95 Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin; Mavrangelos C et al.; The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material . We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp . Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression . The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent . Protein Expr Purif, 2001 Nov, 23(2), 270 - 81 An enhanced and scalable process for the purification of SIV Gag-specific MHC tetramer; Grimm KM et al.; A recently developed method for the identification and quantitation of antigen-specific T lymphocytes involves the use of complexes of biotinylated major histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group . This complex, dubbed the "tetramer," binds to antigen-specific T lymphocytes in vitro, which can then be sorted and counted by fluorescence-activated flow cytometry to measure immune response . Our research has focused on developing the purification process for preparing tetramer reagent . Our goal was to reengineer a published lab-scale purification process to reduce the number of processing steps and to make the process scalable . In our reengineered process, recombinant MHC alpha chain is isolated from Escherichia coli as inclusion bodies by tangential flow filtration . The purified MHC alpha chain is refolded with beta-2-microglobulin and the target peptide antigen to form the class I MHC . The resulting MHC is purified by hydrophobic interaction chromatography (HIC) and biotinylated enzymatically, and the biotinylated MHC is purified by a second HIC step . The tetramer is prepared by mixing biotinylated MHC with an avidin-fluorophore conjugate . The tetramer is further purified to remove any excess MHC or avidin components . Analysis by flow cytometry confirmed that the tetramers generated by this new process gave bright staining and specific binding to CD3+/CD8+ cells of vaccinated monkeys and led to results that were equivalent to those generated with tetramer produced by the original process . Protein Expr Purif, 2001 Nov, 23(2), 219 - 25 Overproduction, purification, and characterization of beta-1,3-glucanase type II in Escherichia coli; Salazar O et al.; An Escherichia coli recombinant system produced soluble and full-length beta-1,3-glucanase type II (BglII) cloned from the yeast-lytic actinomycete Oerskovia xanthineolytica . The expression system was designed to produce recombinant BglII with a six-histidine peptide fused to the carboxy end of the protein . The expression level was optimized to produce 30% of total protein of E . coli as the recombinant protein, releasing 75% to the extracellular space . The 43-kDa recombinant protein was purified by IMAC to homogeneity and its molecular and biochemical characteristics were studied, showing that there are no important functional differences with those properties described for the BglII purified from O . xanthineolytica . Metab Eng, 2001 Oct, 3(4), 362 - 79 The organization of metabolic reaction networks . III . Application for diauxic growth on glucose and lactose; Kremling A et al.; A mathematical model to describe carbon catabolite repression in Escherichia coli is developed and in part validated . The model is aggregated from two functional units describing glucose and lactose transport and degradation . Both units are members of the crp modulon and are under control of a global signal transduction system which calculates the signals that turn on or off gene expression for the specific enzymes . Using isogenic mutant strains, our model is validated by a set of experiments . In these experiments, substrate composition of the preculture and of the experimental culture are varied in order to stimulate the system in different ways . With the obtained measurements (three states in the liquid phase and one intracellular component) a part of the model parameters could be estimated . Therefore all experiments could be sufficiently described with a single set of parameters . Metab Eng, 2001 Oct, 3(4), 313 - 21 Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization; Smolke CD et al.; A synthetic operon containing the crtI and crtY genes, encoding the phytoene desaturase and the lycopene cyclase, respectively, was placed under the control of the araBAD promoter . DNA cassettes encoding mRNA secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the genes . This construct was transformed into Escherichia coli cells harboring the genes for phytoene production . By varying the mRNA secondary structures, we were able to modulate the flux through the carotenoid pathway, resulting in a 300-fold variation in the production of beta-carotene relative to lycopene . In addition, intermediates in the pathway from phytoene to beta-carotene production that are not observed in cells expressing the recombinant operon were observed when the engineered operons were used, indicating that changes in levels of the enzymes affected the formation of intermediates . These results indicate that it is possible to coordinately regulate the genes encoding the enzymes of a metabolic pathway and balance the production of the intermediates . J Mol Biol, 2001 Oct 26, 313(3), 583 - 92 Crystal structure of the Escherichia coli RNA degradosome component enolase; Kuhnel K et al.; The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A . There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules, flexible loops close onto the active site . This closure mimics the conformation of the substrate-bound intermediate . A comparison of the structure of the E . coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme . The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength . The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a specific role to it, a structural role is possible . J Mol Biol, 2001 Oct 26, 313(3), 539 - 57 Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA; Cloutier JF et al.; The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer . Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify . Therefore, we use the NNK analogue 4-{(acetoxymethyl)nitrosamino}-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR) . NNKOAc induced single-strand breaks in a concentration-dependent manner . Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA . Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure . NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine . In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation . Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248 . We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment . Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV . These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA . The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks . These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds . J Mol Biol, 2001 Oct 26, 313(3), 501 - 10 Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli; Balakrishnan L et al.; A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex . This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment . HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59) . Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase . The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC . Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate . The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential . Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function . The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it . In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e . TolC recruitment was not triggered . Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex . J Mol Biol, 2001 Oct 26, 313(3), 485 - 99 The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor; Newman JD et al.; The Rhodobacter sphaeroides extra cytoplasmic function sigma factor, sigma(E), directs transcription of promoters for the cycA gene (cycA P3) and the rpoEchrR operon (rpoE P1) . These genes encode the periplasmic electron carrier cytochrome c(2) and sigma(E)/ChrR, respectively . Using in vitro transcription assays with purified R . sphaeroides core RNA polymerase and sigma(E), we show that ChrR is sufficient to inhibit sigma(E)-dependent transcription . Inhibition is proposed to proceed through a binding interaction, since sigma(E) and ChrR form a 1:1 complex that can be purified when expressed at high levels in Escherichia coli . Active preparations of ChrR and the sigma(E)/ChrR complex each contain stoichiometric zinc . Removal of zinc from ChrR or a single amino acid substitution that abolishes zinc binding, results in a protein that is incapable of inhibiting sigma(E) activity or forming a complex with the sigma factor, indicating that metal binding is important to ChrR activity . Treatment of ChrR with the thiol-modifying reagent p-hydroxymecuriphenylsulfonic acid results in the release of about one mole of zinc per mole of protein . Furthermore, two N-terminal cysteine residues are protected from reaction with the thiol-specific reagent dithionitrobenzoic acid until zinc is removed, suggesting that these residues may be involved in zinc binding . These data indicate that ChrR is a specific anti-sigma factor of sigma(E) that requires zinc for function . Based on amino acid sequence similarity, we propose that ChrR is part of a family of similar anti-sigma factors that are found in alpha and gamma proteobacteria . Biochem Biophys Res Commun, 2001 Nov 2, 288(3), 499 - 502 Hydrolysis of oxidized nucleotides by the Escherichia coli Orf135 protein; Kamiya H et al.; To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides . Of the nucleotides tested, 2-hydroxydeoxyadenosine 5'-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5'-triphosphate, were hydrolyzed by this protein . These damaged deoxynucleotides elicit transversion mutations in E . coli (Inoue, M., Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, H . (1998) J . Biol . Chem . 273, 11069-11074) . These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides . Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12724 - 9 Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway; Hubner A et al.; RpoS and RpoN are two alternative sigma factors typically associated with general stress responses in bacteria . To date, there has been no experimental evidence that RpoS and RpoN can directly control the expression of one another . Herein, using a combined strategy of gene disruption and genetic complementation targeting rpoN and rpoS in Borrelia burgdorferi strain 297, we describe a regulatory network for B . burgdorferi . In this network, RpoN controls the expression of RpoS, which, in turn, governs the expression of two important membrane lipoproteins, outer surface protein C and decorin-binding protein A, and likely other proteins of B . burgdorferi . Our findings provide a foundation for elucidating further key regulatory networks that potentially impact many aspects of B . burgdorferi's parasitic strategy, host range, and virulence expression. Anal Biochem, 2001 Nov 1, 298(1), 57 - 61 An FKBP12 binding assay based upon biotinylated FKBP12; Carreras CW et al.; A binding assay was developed for measuring the affinity of FKBP12 ligands . A biotinylation signal sequence was fused to the 5' end of the human FKBP12 gene, and the fusion protein was expressed in Escherichia coli with biotin ligase . The fusion protein was immobilized in avidin-coated multiwell plates, and varying concentrations of test ligands were allowed to compete with {3H}FK506 for FKBP12 sites on the plate . The assay provided Kd values for FK520, 32-hydroxyethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values . The assay provides a convenient and rapid method for the assessment of FKBP12 binding by small molecules . Arch Biochem Biophys, 2001 Nov 1, 395(1), 57 - 68 A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function; Scott EE et al.; The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility . Histidine residues were appended to the C-terminus to simplify purification . The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column . Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate . All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation . These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis . Arch Biochem Biophys, 2001 Nov 1, 395(1), 25 - 31 Random mutagenesis of human cytochrome p450 2A6 and screening with indole oxidation products; Nakamura K et al.; Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism . SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating . One mutant, F209T, showed indole 3-hydroxylation <WT but had a k(cat) for coumarin 7-hydroxylation 13-fold >WT . The double mutant L240C/N297Q consistently produced very blue colonies . Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products . When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants . This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products . Arch Biochem Biophys, 2001 Nov 1, 395(1), 14 - 20 Effects of lipids on the interaction of SecA with model membranes; Ahn T et al.; The effects of nonlamellar-prone lipids, diacylglycerol and phosphatidylethanolamine (PE), on the kinetic association of SecA with model membranes were examined by measuring changes in the intrinsic emission fluorescence with a stopped-flow apparatus . Upon interaction with standard liposomes composed of 50 mol% dioleolyphosphatidylcholine (DOPC) and 50 mol% of dioleoylphosphatidylglycerol (DOPG), the intrinsic fluorescence intensity of SecA was decreased after a lapse of time with a rate constant of 0.0049 s(-1) . When the DOPC of the standard vesicles was gradually replaced with either dioeloyl PE (DOPE) or Escherichia coli (E . coli) PE, the rate constant increased appreciably as a function of PE concentration, in the order DOPE > E . coli PE . In addition, when the PE of E . coli PE/DOPG (50/50) vesicles was replaced with more than 5 mol% dioleoylglycerol (DOG), the rate constant further increased by 40% . The incorporation of nonlamellar-prone lipids in the vesicles also enhanced the binding of SecA to model membranes in the order DOPE > or = E . coli PE/DOG > E . coli PE > DOPC . These results provide the first kinetic evidence for the importance of nonlamellar-prone phospholipids for the association rate of SecA with membranes . J Immunol, 2001 Nov 1, 167(9), 5470 - 7 Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen; Binder M et al.; IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals . We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest . Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P . interpunctella larvae . cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated . Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography . Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions . It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients' IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel . Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens . Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals. J Immunol, 2001 Nov 1, 167(9), 5273 - 7 Immunization of mice against West Nile virus with recombinant envelope protein; Wang T et al.; West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis . Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli . The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments . Patients with WN virus infection had Abs that recognized the recombinant E protein . C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus . Passive administration of E protein antisera was also sufficient to afford immunity . E protein is a candidate vaccine to prevent WN virus infection. J Immunol, 2001 Nov 1, 167(9), 5202 - 8 The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r; Kardos J et al.; The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex . Activated C1r then cleaves and activates zymogen C1s . C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity . The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP) . To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli . We successfully renatured the inclusion body proteins . After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other . To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed . Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain . We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s . Therefore, we propose that CCP2 module provides accessory binding sites . Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain . Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure . Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation. J Immunol, 2001 Nov 1, 167(9), 5129 - 35 Structural analysis of mutants of high-affinity and low-affinity p-azophenylarsonate-specific antibodies generated by alanine scanning of heavy chain complementarity-determining region 2; Parhami-Seren B et al.; Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71 . Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity . Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used . All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain . However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities . When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65 . These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding. J Immunol, 2001 Nov 1, 167(9), 5067 - 76 Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo; Pulendran B et al.; The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood . In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens . We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling . Coinjections of E . coli LPS + OVA or P . gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles . E . coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5 . In contrast, P . gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma . Consistent with these results, E . coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P . gingivalis LPS did not . Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha . Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E . coli LPS, but not P . gingivalis LPS . Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo. J Bacteriol, 2001 Nov, 183(22), 6721 - 5 Colicin A immunity protein interacts with the hydrophobic helical hairpin of the colicin A channel domain in the Escherichia coli inner membrane; Nardi A et al.; The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA) . This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai) . We constructed a series of fusion proteins in which various numbers of sp-pfColA alpha-helices were fused to alkaline phosphatase (AP) . We showed that a fusion protein made up of the hydrophobic alpha-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells . This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin. J Bacteriol, 2001 Nov, 183(22), 6684 - 7 The dimerization function of MinC resides in a structurally autonomous C-terminal domain; Szeto TH et al.; Limited proteolysis of the Escherichia coli cell division inhibitor MinC reveals that its dimerization function resides in a structurally autonomous C-terminal domain . We show that cytoplasmic MinC is poised near the monomer-dimer equilibrium and propose that it only becomes entirely dimeric once recruited to the membrane by MinD. J Bacteriol, 2001 Nov, 183(22), 6645 - 53 Screening of environmental DNA libraries for the presence of genes conferring Na(+)(Li(+))/H(+) antiporter activity on Escherichia coli: characterization of the recovered genes and the corresponding gene products; Majernik A et al.; Environmental DNA libraries prepared from three different soils were screened for genes conferring Na(+)(Li(+))/H(+) antiporter activity on the antiporter-deficient Escherichia coli strain KNabc . The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl . Two positive E . coli clones were obtained during the initial screening of 1,480,000 recombinant E . coli strains . Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li(+)-resistant phenotype . The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na(+)/H(+) antiporter belonging to the NhaA family . The insert of pAM3 harbored the DNA region of E . coli K-12 containing nhaA, nhaR, and gef . This region is flanked by highly conserved insertion elements . The sequence identity with E . coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E . coli . The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E . coli and enabled the antiporter-deficient E . coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0 . The Na(+)/H(+) antiporter activity in everted membrane vesicles that were derived from the E . coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5 . The maximal activity was observed at pHs 8.3 and 8.6, respectively . The K(m) values of both antiporters for Na(+) were approximately 10-fold higher than the values for Li(+). J Bacteriol, 2001 Nov, 183(22), 6538 - 42 Deletion of lolB, encoding an outer membrane lipoprotein, is lethal for Escherichia coli and causes accumulation of lipoprotein localization intermediates in the periplasm; Tanaka K et al.; Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane . In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex . On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo . To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene . At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp . LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane . Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E . coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking. J Bacteriol, 2001 Nov, 183(22), 6532 - 7 Involvement of the N terminus of ribosomal protein L11 in regulation of the RelA protein of Escherichia coli; Yang X et al.; Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes . The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA . To investigate the role of L11 in the stringent response, a derivative of rplK encoding L11 lacking the N-terminal 36 amino acids (designated 'L11) was constructed . Bacteria overexpressing 'L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation . In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response . The overexpressed 'L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA . By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11 . To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis . The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype . These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA. Thromb Res, 2001 Aug 1, 103(3), 165 - 72 Thrombotic events revisited in children with acute lymphoblastic leukemia: impact of concomitant Escherichia coli asparaginase/prednisone administration; Nowak-Gottl U et al.; Recently published data suggest that the prothrombin G20210A variant, the TT677 methylenetetrahydrofolate reductase genotype, the factor V G1691A mutation, deficiencies of protein C, protein S, antithrombin, and elevated lipoprotein (a) concentrations were associated with venous thromboembolism in childhood patients treated according to the BFM protocol . To unravel the role of these prothrombotic risk factors and different treatment modalities, the present comparative study was performed in childhood leukemia patients of the same living population . Four hundred and twenty consecutively recruited leukemic children (BFM n=300; COALL n=120) were enrolled in this study with respect to the presence of prothrombotic risk factors and the occurrence of symptomatic venous thrombosis . No significant difference was found in the prevalence rates of thrombotic risk factors in the Caucasian populations studied . Symptomatic venous thromboembolism occurred in 11.6% of BFM patients compared with 2.5% in the COALL treatment group {odds ratio (OR)/95% confidence intervals (CI): 7.7/1.8-32.6; P=.005} . Including age, prothrombotic risk factors, central venous lines, treatment protocols, and anti-leukemic drugs in a logistic regression model, only the concomitant Escherichia coli asparaginase/prednisone administration in leukemic children suffering from a prothrombotic risk factor was found to increase the rate of thrombotic manifestations during leukemia treatment in patients of the same Caucasian origin (OR/95% CI: 34.5/4.39-271.42; P=.0008) . Based on the data presented here, we suggest the use of prednisone and E . coli asparaginase concomitantly administered in a leukemic patient suffering from a prothrombotic risk factor to be responsible for the onset of venous thrombosis in the majority of cases. Cell, 2001 Oct 19, 107(2), 235 - 46 GroEL/GroES-mediated folding of a protein too large to be encapsulated; Chaudhuri TK et al.; The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered . We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria . We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release . Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution . Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES. Biochem J, 2001 Nov 1, 359(Pt 3), 507 - 16 Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases; Jowsey IR et al.; GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date . Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography . The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2) . Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities . The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme . Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively) . Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences . The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow . Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species . The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken. Biochem J, 2001 Nov 1, 359(Pt 3), 497 - 505 Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A; Himpel S et al.; Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr) . In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2 . Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells . Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated . When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321 . A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A . MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A . Enzymic activity was not affected in the DYRK1A-Y111F mutant . The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop. Inorg Chem, 1997 May 7, 36(10), 2079 - 2083 Methyl Transfer to Mercury Thiolates: Effects of Coordination Number and Ligand Dissociation; Wilker JJ et al.; The complexes {(CH(3))(4)N}(2){Hg(SC(6)H(5))(4)} and {(C(4)H(9))(4)N}{Hg(SC(6)H(5))(3)} demethylate (CH(3)O)(3)PO as revealed by (1)H, (31)P{(1)H}, and (199)Hg{(1)H} NMR spectroscopy in DMSO-d(6) solution . The products of the {(CH(3))(4)N}(2){Hg(SC(6)H(5))(4)} reaction are CH(3)SC(6)H(5), (CH(3)O)(2)PO(2)(-), and {Hg(SC(6)H(5))(3)}(-), whereas {Hg(SC(6)H(5))(3)}(-) demethylates (CH(3)O)(3)PO to yield CH(3)SC(6)H(5) and {Hg(SC(6)H(5))(2){(CH(3)O)(2)PO(2)}}(-) . Kinetic and solution studies of {(CH(3))(4)N}(2){Hg(SC(6)H(5))(4)} reveal a rapid equilibrium between bound and free thiolate . The dissociated thiolate is the nucleophile active toward (CH(3)O)(3)PO . These results imply that the metal center of the inactive mercury derivative of the Escherichia coli Ada DNA alkylation repair protein may comprise a three-coordinate {Hg(S-cysteine)(3)}(-) moiety and an unbound, protonated cysteine (HS-Cys69). Inorg Chem, 1997 Mar 12, 36(6), 969 - 978 Alkyl Transfer to Metal Thiolates: Kinetics, Active Species Identification, and Relevance to the DNA Methyl Phosphotriester Repair Center of Escherichia coli Ada; Wilker JJ et al.; The Ada protein of Escherichia coli employs a {Zn(S-cys)(4)}(2)(-) site to repair deoxyribonucleic acid alkyl phosphotriester lesions . The alkyl group is transferred to a cysteine thiolate in a stoichiometric reaction . We describe a functional model for this chemistry in which a thiolate of {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} accepts a methyl group from (CH(3)O)(3)PO . The thiolate salt (CH(3))(4)N(SC(6)H(5)) is also active in methyl transfer, but the thiol C(6)H(5)SH fails to react . Conductivity measurements and kinetic studies demonstrate that {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} forms ion pairs in dimethyl sulfoxide (DMSO) solution (K(IP) = 13 +/- 4 M(-)(1)) which exhibit diminished reactivity . The reaction of {Zn(SC(6)H(5))(4)}(2)(-) with (CH(3)O)(3)PO is first order with respect to each reagent . A second-order rate constant for this reaction, k(Zn), was determined to be (1.6 +/- 0.3) x 10(-)(2) M(-)(1) s(-)(1) . From kinetic data and equilibria studies, all reactivity of {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} toward (CH(3)O)(3)PO could be attributed to dissociated thiolate . Metal complexes representing alternative protein sites were prepared and displayed the following kinetic trend of methyl transfer ability: {(CH(3))(4)N}(2){Zn(SC(6)H(5))(4)} > {(CH(3))(4)N}(2){Co(SC(6)H(5))(4)} approximately {(CH(3))(4)N}(2){Cd(SC(6)H(5))(4)} > {(CH(3))(4)N}{Zn(SC(6)H(5))(3)(MeIm)} > {Zn(SC(6)H(5))(2)(MeIm)(2)}, where MeIm = 1-methylimidazole . These results are consistent with a dissociated thiolate being the active species and suggest that a similar mechanism might apply to alkyl phosphotriester repair by Ada. Biochemistry, 2001 Oct 30, 40(43), 13079 - 87 Novel insights into the basis for Escherichia coli superoxide dismutase's metal ion specificity from Mn-substituted FeSOD and its very high E(m); Vance CK et al.; Fe and Mn are both entrained to the same chemical reaction in apparently superimposable superoxide dismutase (SOD) proteins . However, neither Fe-substituted MnSOD nor Mn-substituted FeSOD is active . We have proposed that the two SOD proteins must apply very different redox tuning to their respective metal ions and that tuning appropriate for one metal ion results in a reduction potential (E(m)) for the other metal ion that is either too low (Fe) or too high (Mn) {Vance and Miller (1998) J . Am . Chem . Soc . 120, 461-467} . We have demonstrated that this is true for Fe-substituted MnSOD from Escherichia coli and that this metal ion-protein combination retains the ability to reduce but not oxidize superoxide . We now demonstrate that the corollary is also true: Mn-substituted FeSOD {Mn(Fe)SOD} has a very high E(m) . Specifically, we have measured the E(m) of E . coli MnSOD to be 290 mV vs NHE . We have generated Mn(Fe)SOD and find that Mn is bound in an environment similar to that of the native (Mn)SOD protein . However, the E(m) is greater than 960 mV vs NHE and much higher than MnSOD's E(m) of 290 mV . We propose that the different tuning stems from different hydrogen bonding between the proteins and a molecule of solvent that is coordinated to the metal ion in both cases . Because a proton is taken up by SOD upon reduction, the protein can exert very strong control over the E(m), by modulating the degree to which coordinated solvent is protonated, in both oxidation states . Thus, coordinated solvent molecules may have widespread significance as "adapters" by which proteins can control the reactivity of bound metal ions. Biochemistry, 2001 Oct 30, 40(43), 13068 - 78 Zinc-thiolate intermediate in catalysis of methyl group transfer in Methanosarcina barkeri; Gencic S et al.; Methyl group transfer reactions are essential in methane-forming pathways in all methanogens . The involvement of zinc in catalysis of methyl group transfer was studied for the methyltransferase enzyme MT2-A important for methanogenesis in Methanosarcina barkeri growing on methylamines . Zinc was shown to be required for MT2-A activity and was tightly bound by the enzyme with an apparent stability constant of 10(13.7) at pH 7.2 . Oxidation was a factor influencing activity and metal stoichiometry of purified MT2-A preparations . Methods were developed to produce inactive apo MT2-A and to restore full activity with stoichiometric reincorporation of Zn(2+) . Reconstitution with Co(2+) yielded an enzyme with 16-fold higher specific activity . Cysteine thiolate coordination in Co(2+)-MT2-A was indicated by high absorptivity in the 300-400 nm charge transfer region, consistent with more than one thiolate ligand at the metal center . Approximate tetrahedral geometry was indicated by strong d-d transition absorbance centered at 622 nm . EXAFS analyses of Zn(2+)-MT2-A revealed 2S + 2N/O coordination with evidence for involvement of histidine . Interaction with the substrate CoM (2-mercaptoethanesulfonic acid) resulted in replacement of the second N/O group with S, indicating direct coordination of the CoM thiolate . UV-visible spectroscopy of Co(2+)-MT2-A in the presence of CoM also showed formation of an additional metal-thiolate bond . Binding of CoM over the range of pH 6.2-7.7 obeyed a model in which metal-thiolate formation occurs separately from H(+) release from the enzyme-substrate complex . Proton release to the solvent takes place from a group with apparent pK(a) of 6.4, and no evidence for metal-thiolate protonation was found . It was determined that substrate metal-thiolate bond formation occurs with a Delta G degrees ' of -6.7 kcal/mol and is a major thermodynamic driving force in the overall process of methyl group transfer. Biochemistry, 2001 Oct 30, 40(43), 13060 - 7 Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase; Chih HW et al.; Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase . The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding . This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product . Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction . The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate . The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction . The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes. Biochemistry, 2001 Oct 30, 40(43), 13051 - 9 Targeted disulfide cross-linking of the MotB protein of Escherichia coli: evidence for two H(+) channels in the stator Complex; Braun TF et al.; Bacterial flagella are turned by rotary motors that obtain energy from the membrane gradient of protons or sodium ions . The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes that contain multiple copies of each protein . The complexes conduct ions across the membrane, and couple ion flow to motor rotation by a mechanism that appears to involve conformational changes {Kojima, S., and Blair, D . F . (2001) Biochemistry 40, 13041-13050} . Structural information on the MotA/MotB complex is very limited . MotA has four membrane-spanning segments, and MotB has one . We have begun a targeted disulfide-cross-linking study to probe the arrangement of membrane segments in the MotA/MotB complex, beginning with the single membrane segment of MotB . Cys residues were introduced in 21 consecutive positions in the segment, and disulfide cross-linking was studied in MotA/MotB complexes either in membranes or detergent solution . Most of the Cys-substituted MotB proteins formed disulfide-linked dimers in significant yield upon oxidation . The yield of dimer varied regularly with the position of the Cys substitution, following the pattern expected for a parallel, symmetric dimer of alpha-helices . In a structural model based on the cross-linking experiments, critical Asp32 residues that are believed to facilitate proton movement are positioned on separate surfaces of the MotB dimer and so probably function within two distinct proton channels . Regions accessible to solvent were mapped by measuring the reactivity of introduced Cys residues toward N-ethyl maleimide and a charged methanethiosulfonate reagent . Positions near the middle of the segment were inaccessible to sulhydryl reagents . Positions within 6-8 residues of either end, which includes residues around Asp32, were accessible. Biochemistry, 2001 Oct 30, 40(43), 13015 - 9 The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli; Sahin-Toth M et al.; Binding specificity in lactose permease toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by nonspecific hydrophobic interactions with the non-galactosyl moiety {Sahin-Toth, M., Akhoon, K . M., Runner, J., and Kaback, H . R . (2000) Biochemistry 39, 5097-5103} . To characterize the contribution of individual hydroxyls, binding of structural analogues of p-nitrophenyl alpha-D-galactopyranoside (NPG) was examined by site-directed N-{(14)C}ethylmaleimide (NEM) labeling of the substrate-protectable Cys148 in the binding site . NPG blocks NEM alkylation of Cys148 with an apparent affinity of approximately 14 microM . A deoxy derivative at position C-2 binds with 25-fold lower affinity (K(D) 0.35 mM), and the deoxy analogue at C-3 exhibits ca . 70-fold decreased binding (K(D) 1 mM), while binding of 6-deoxy-NPG is at least 130-fold diminished (K(D) 1.9 mM) . Remarkably, the C-4 deoxy derivative of NPG binds with almost 1500-fold reduced affinity (K(D) approximately 20 mM) . No significant substrate protection is afforded by NPG analogues with methoxy (CH(3)-O-) substitutions at positions C-3, C-4, and C-6 . In contrast, the C-2 methoxy analogue binds almost normally (K(D) 26 microM) . The results confirm and extend the observations that the C-2, C-3, C-4, and C-6 OH groups of galactopyranosides participate in important H-bonding interactions . Moreover, the C-4 hydroxyl is identified as the major determinant of ligand binding, suggesting that sugar recognition in lactose permease may have evolved to discriminate primarily between gluco- and galactopyranosides. Biochemistry, 2001 Oct 30, 40(43), 12967 - 73 Physical properties of human polynucleotide kinase: hydrodynamic and spectroscopic studies; Mani RS et al.; Human polynucleotide kinase (hPNK) is a putative DNA repair enzyme in the base excision repair pathway required for processing and rejoining strand-break termini . This study represents the first systematic examination of the physical properties of this enzyme . The protein was produced in Escherichia coli as a His-tagged protein, and the purified recombinant protein exhibited both the kinase and the phosphatase activities . The predicted relative molecular mass (M(r)) of the 521 amino acid polypeptide encoded by the sequenced cDNA for PNK and the additional 21 amino acids of the His tag is 59,538 . The M(r) determined by low-speed sedimentation equilibrium under nondenaturing conditions was 59,600 +/- 1000, indicating that the protein exists as a monomer, in contrast to T4 phage PNK, which exists as a homotetramer . The size and shape of hPNK in solution were determined by analytical ultracentrifugation studies . The protein was found to have an intrinsic sedimentation coefficient, s(0)(20,w), of 3.54 S and a Stokes radius, R(s), of 37.5 A . These hydrodynamic data, together with the M(r) of 59 600, suggest that hPNK is a moderately asymmetric protein with an axial ratio of 5.51 . Analysis of the secondary structure of hPNK on the basis of circular dichroism spectra, which revealed the presence of two negative dichroic bands located at 218 and 209 nm, with ellipticity values of -7200 +/- 300 and -7800 +/- 300 deg x cm(2) x d(mol(-1), respectively, indicated the presence of approximately 50% beta-structure and 25% alpha-helix . Binding of ATP to the protein induced an increase in beta-structure and perturbed tryptophan, tyrosine, and phenylalanine signals observed by aromatic CD and UV difference spectroscopy. Biochemistry, 2001 Oct 30, 40(43), 12904 - 12 Steady-state and pre-steady-state kinetic analysis of Mycobacterium tuberculosis pantothenate synthetase; Zheng R et al.; Pantothenate synthetase (EC 6.3.2.1), encoded by the panC gene, catalyzes the essential ATP-dependent condensation of D-pantoate and beta-alanine to form pantothenate in bacteria, yeast and plants . Pantothenate synthetase from Mycobacterium tuberculosis was expressed in E . coli, purified to homogeneity, and found to be a homodimer with a subunit molecular mass of 33 kDa . Initial velocity, product, and dead-end inhibition studies showed the kinetic mechanism of pantothenate synthetase to be Bi Uni Uni Bi Ping Pong, with ATP binding followed by D-pantoate binding, release of PP(i), binding of beta-alanine, followed by the release of pantothenate and AMP . Michaelis constants were 0.13, 0.8, and 2.6 mM for D-pantoate, beta-alanine, and ATP, respectively, and the turnover number, k(cat), was 3.4 s(-1) . The formation of pantoyl adenylate, suggested as a key intermediate by the kinetic mechanism, was confirmed by (31)P NMR spectroscopy of {(18)O}AMP produced from (18)O transfer using {carboxyl-(18)O}pantoate . Single-turnover reactions for the formation of pyrophosphate and pantothenate were determined using rapid quench techniques, and indicated that the two half-reactions occurred with maximum rates of 1.3 +/- 0.3 and 2.6 +/- 0.3 s(-)(1), respectively, consistent with pantoyl adenylate being a kinetically competent intermediate in the pantothenate synthetase reaction . These data also suggest that both half-reactions are partially rate-limiting . Reverse isotope exchange of {(14)C}-beta-alanine into pantothenate in the presence of AMP was observed, indicating the reversible formation of the pantoyl adenylate intermediate from products. Biochemistry, 2001 Oct 30, 40(43), 12896 - 903 Synthesis and biochemical characterization of a phosphorylated analogue of the response regulator CheB; Saxl RL et al.; CheB is a response regulator protein in the bacterial chemotaxis two-component signal transduction pathway . Methylesterase CheB functions together with methyltransferase CheR to modulate the level of glutamate methylation in transmembrane chemoreceptors in response to environmental stimuli . The level of glutamate methylation in turn indirectly controls the direction of flagellar rotation . Like most two-component response regulators, CheB is activated in vivo by phosphorylation of a single aspartate, Asp 56, in its regulatory domain . Extensive biochemical and crystallographic studies have been completed on the inactive, unphosphorylated form of CheB . Because of the inherent lability of aspartyl phosphate bonds and the intrinsic phosphatase activity of CheB, the activated, phosphorylated form of CheB cannot be isolated for further characterization . We present a synthetic scheme to prepare an analogue of phosphorylated CheB using site-specific mutagenesis and chemical modification strategies . Initially, the two native cysteines found in CheB were substituted by serines and a cysteine was substituted for Asp 56 to yield D56C/C207S/C309S CheB . The unique cysteine in the substituted form of CheB was modified by sodium thiophosphate, Na(3)SPO(3), using two sequential disulfide bond exchange reactions . The analogue, D56C/C207S/C309S CheB-SPO(3), contained a thiophosphate group covalently bonded to the protein through a disulfide linkage at residue 56 . Mass spectrometry showed that the protein was singly modified . Reverse phase chromatography showed that greater than 95% of the protein was modified under optimized conditions and that the analogue had a half-life of 28 days . In in vitro methylesterase assays in the presence of Mg(2+), the analogue exhibited activity equivalent to that of fully phosphorylated C207S/C309S CheB . Thus, D56C/C207S/C309S CheB-SPO(3) is a stable analogue that may be useful for characterization of the active form of CheB. Biochemistry, 2001 Oct 30, 40(43), 12826 - 32 Structures of tetrahydrobiopterin binding-site mutants of inducible nitric oxide synthase oxygenase dimer and implicated roles of Trp457; Aoyagi M et al.; To better understand potential roles of conserved Trp457 of the murine inducible nitric oxide synthase oxygenase domain (iNOS(ox); residues 1-498) in maintaining the structural integrity of the (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) binding site located at the dimer interface and in supporting H(4)B redox activity, we determined crystallographic structures of W457F and W457A mutant iNOS(ox) dimers (residues 66-498) . In W457F iNOS(ox), all the important hydrogen-bonding and aromatic stacking interactions that constitute the H(4)B binding site and that bridge the H(4)B and heme sites are preserved . In contrast, the W457A mutation results in rearrangement of the Arg193 side chain, orienting its terminal guanidinium group almost perpendicular to the ring plane of H(4)B . Although Trp457 is not required for dimerization, both Trp457 mutations led to the increased mobility of the N-terminal H(4)B binding segment (Ser112-Met114), which might indicate reduced stability of the Trp457 mutant dimers . The Trp457 mutant structures show decreased pi-stacking with bound pterin when the wild-type pi-stacking Trp457 position is occupied with the smaller Phe457 in W457F or positive Arg193 in W457A . The reduced pterin pi-stacking in these mutant structures, relative to that in the wild-type, implies stabilization of reduced H(4)B and destabilization of the pterin radical, consequently slowing electron transfer to the heme ferrous-dioxy (Fe(II)O(2)) species during catalysis . These crystal structures therefore aid elucidation of the roles and importance of conserved Trp457 in maintaining the structural integrity of the H(4)B binding site and of H(4)B-bound dimers, and in influencing the rate of electron transfer between H(4)B and heme in NOS catalysis. Biochemistry, 2001 Oct 30, 40(43), 12808 - 18 Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential; Murray JM et al.; Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylalanine (TPQ) cofactor from an active site tyrosine by a single turnover mechanism; second, the oxidative deamination of primary amine substrates with the production of aldehyde, hydrogen peroxide, and ammonia catalyzed by the mature enzyme . The importance of active site residues in both of these processes has been investigated by structural studies and site-directed mutagenesis in enzymes from various organisms . One conserved residue is a tyrosine, Tyr369 in the Escherichia coli enzyme, whose hydroxyl is hydrogen bonded to the O4 of TPQ . To explore the importance of this site, we have studied a mutant enzyme in which Tyr369 has been mutated to a phenylalanine . We have determined the X-ray crystal structure of this variant enzyme to 2.1 A resolution, which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme . Reaction of the enzyme with the irreversible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the reactivity of Y369F compared with wild type with more efficient formation of an adduct (lambda(max) = 525 nm) perhaps reflecting increased mobility of the TPQ adduct within the active site of Y369F . Titration with 2-HP also reveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is not essential for TPQ formation, although we have not measured the rate of TPQ biogenesis . The UV-vis spectrum of the Y369F protein shows a broader peak and red-shifted lambda(max) at 496 nm compared with wild type (480 nm), consistent with an altered electronic structure of TPQ . Steady-state kinetic measurements reveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the K(M) for substrate beta-phenethylamine increases significantly, apparently due to an elevated pK(a) (5.75-6.5) for the catalytic base, Asp383, that should be deprotonated for efficient binding of protonated substrate . At pH 7.0, the K(M) for wild type and Y369F are similar at 1.2 and 1.5 microM, respectively, while k(cat) is decreased from 15 s(-1) in wild type to 0.38 s(-1), resulting in a 50-fold decrease in k(cat)/K(M) for Y369F . Transient kinetics experiments indicate that while the initial stages of enzyme reduction are slower in the variant, these do not represent the rate-limiting step . Previous structural and solution studies have implicated Tyr369 as a component of a proton shuttle from TPQ to dioxygen . The moderate changes in kinetic parameters observed for the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can be compensated for efficiently within the active site. Biochemistry, 2001 Oct 30, 40(43), 12801 - 7 Folding determinants of LDL receptor type A modules; Koduri V et al.; To investigate how three disulfide bonds and coordination of a calcium ion cooperate to specify the structure of an LDL-A module, we studied the interdependence of disulfide bond formation and calcium coordination in the folding of ligand-binding module 5 of the LDL receptor (LR5) . In variants of LR5 containing only a single pair of cysteines normally disulfide-bonded in the native polypeptide, the addition of calcium does not alter the effective concentration of one cysteine for the other . LR5 only exhibits a calcium-dependent preference for formation of native disulfide bonds and detectable calcium-induced changes in structure when the two C-terminal disulfide bonds are present . Furthermore, when the conformation of this two-disulfide variant of LR5 is probed by NMR in the presence of calcium, only the C-terminal lobe of the module, which contains the calcium coordination site, acquires a near-native conformation; the N-terminal lobe appears to be disordered . These findings contrast with studies of other model proteins, like BPTI, in which formation of a single disulfide bond is sufficient to drive the entire domain to acquire a stable, nativelike fold. Biochemistry, 2001 Oct 30, 40(43), 12772 - 81 Structure of beta-ketoacyl-{acyl carrier protein} reductase from Escherichia coli: negative cooperativity and its structural basis; Price AC et al.; The structure of beta-ketoacyl-{acyl carrier protein} reductase (FabG) from Escherichia coli was determined via the multiwavelength anomalous diffraction technique using a selenomethionine-labeled crystal containing 88 selenium sites in the asymmetric unit . The comparison of the E . coli FabG structure with the homologous Brassica napus FabG.NADP(+) binary complex reveals that cofactor binding causes a substantial conformational change in the protein . This conformational change puts all three active-site residues (Ser 138, Tyr 151, and Lys 155) into their active configurations and provides a structural mechanism for allosteric communication between the active sites in the homotetramer . FabG exhibits negative cooperative binding of NADPH, and this effect is enhanced by the presence of acyl carrier protein (ACP) . NADPH binding also increases the affinity and decreases the maximum binding of ACP to FabG . Thus, unlike other members of the short-chain dehydrogenase/reductase superfamily, FabG undergoes a substantial conformational change upon cofactor binding that organizes the active-site triad and alters the affinity of the other substrate-binding sites in the tetrameric enzyme. Biochemistry, 2001 Oct 30, 40(43), 12761 - 71 (15)N backbone dynamics of ferricytochrome b(562): comparison with the reduced protein and the R98C variant; Assfalg M et al.; The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy . The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed . (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant . In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame {Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C . M., and Barker, P . D . (2000) Biochemistry 39, 1499-1514} . The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species . The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system . Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2 . Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein . The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species . A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562) . In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing . The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant . It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity. Plant Mol Biol, 2001 Nov, 47(4), 519 - 31 Gene isolation and characterization of two acyl CoA oxidases from soybean with broad substrate specificities and enhanced expression in the growing seedling axis; Agarwal AK et al.; The first committed step in the beta-oxidation of fatty acids is catalyzed by the enzyme acyl-CoA oxidase (ACOX), which oxidizes a fatty acyl-CoA to a 2-trans-enoyl-CoA . To understand the role of beta-oxidation during seedling growth in soybean, two ACOX cDNAs were isolated by screening a seedling library with a DNA fragment obtained by RT-PCR by using degenerate oligonucleotides . The two cDNAs (ACX1;1 and ACX1;2) are 86% identical to each other at the nucleotide and the amino acid level . Their deduced amino acid sequences share significant homology with known acyl-CoA oxidases, including the conserved CGGHGY motif, a putative flavin mononucleotide binding site . In both sequences, the last three amino acids, ARL, represent a putative peroxisome targeting signal . The mRNA and protein of both cDNAs accumulated in all seedling tissues, with relatively stronger expression in the growing seedling axis and hypocotyl, and weaker expression in the cotyledon . Immunolocalization studies indicated that the two proteins were localized in the phloem cells of hypocotyl tissue . The two cDNAs were expressed in Escherichia coli and shown to possess acyl-CoA oxidase activity . With fatty acyl-CoA substrates of varying chain lengths, it was demonstrated that both ACX1;1 and ACX1;2 have broad substrate specificities (C8-C18) . The stronger expression of ACX1;1 and ACX 1;2 in the axis and hypocotyl tissue, the weaker expression in the oil-rich cotyledon tissue, and the broad substrate specificities suggest that the two acyl-CoA oxidases might play a general house-keeping role during soybean seedling growth, such as the turnover of membrane lipids. Comb Chem High Throughput Screen, 2001 Nov, 4(7), 545 - 52 Identification of a novel human peroxisomal 2,4-dienoyl-CoA reductase related protein using the M13 phage protein VI phage display technology; Amery L et al.; Recently, we reported the successful use of the gVI-cDNA phage display technology to clone cDNAs coding for novel peroxisomal enzymes by affinity selection using immobilized antisera directed against peroxisomal subfractions (Fransen, M.; Van Veldhoven, P.P.; Subramani, S . Biochem . J., 1999, 340, 561-568) . To identify other unknown peroxisomal enzymes, we further exploited this promising approach . Here we report the isolation and cloning of another novel human cDNA encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal targeting signal (PTS1) . Primary structure analysis revealed that this molecule shared the highest sequence similarity to members of the 2,4-dienoyl-CoA reductase (DCR) family . However, functional analysis indicated that a recombinantly expressed version of the novel protein did not possess DCR activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA as a substrate . The recombinant protein interacted with HsPex5p, the human PTS1-binding protein . Binding was competitively inhibited by a PTS1-containing peptide and was abolished when the last amino acid of the PTS1 signal was deleted . Transfection of mammalian cells with gene fusions between green fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal localization and, therefore, the functionality of the PTS1 . These results further demonstrate the suitability of the gVI-cDNA phage display technology for cDNA expression cloning using an antibody as a probe. Biotechnol Bioeng, 2001 Nov, 76(3), 187 - 92 Towards the development of a minimal cell model by generalization of a model of Escherichia coli: use of dimensionless rate parameters; Browning ST et al.; A model of a minimal cell would be a valuable tool in identifying the organizing principles that relate the static sequence information of the genome to the dynamic functioning of the living cell . Our approach for developing a minimal cell model is to first generalize an existing model of Escherichia coli by expressing reaction rates as ratios to a set of reference parameters . This generalized model is a prototype minimal cell model that will be developed by adding detail to explicitly include each chemical species . We tested the concept of a generalized model by testing the effect of scaling all enzyme-catalyzed reactions in the E . coli model . The scaling has little effect on cellular function for a wide range of kinetic ratios, where the kinetic ratio is defined as the rate of all enzyme-catalyzed reactions in a given model relative to those in the E . coli model . Biotechnol Bioeng, 2001 Nov 20, 75(4), 492 - 4 Thiosulphate improves yield of hydrogen production from glucose by the immobilized formate hydrogenlyase system of Escherichia coli; Nandi R et al.; Escherichia coli cells with formate hydrogenlyase activity that were immobilized in agar beads produced hydrogen from glucose at the approximate yield of 0.6 mole of hydrogen per mole . Succinate or thiosulphate added to glucose increased hydrogen production two-fold . Thiosulphate did not increase the rate of hydrogen production but reduced the consumption of glucose for the same amount of hydrogen produced compared to control . Oxaloacetate and traces of pyruvate instead of succinate accumulated at the end when thiosulphate was present . Biotechnol Bioeng, 2001 Nov 20, 75(4), 439 - 50 Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli; DeLisa MP et al.; Recent reports have shown that bacterial cell-cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, the role of quorum sensing in nonpathogenic E . coli is less clear and, in particular, there is no information regarding the role of quorum sensing in overexpression of plasmid-encoded genes . In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E . coli . Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling . Importantly, our findings demonstrate for the first time that recombinant E . coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway . Biotechnol Bioeng, 2001 Nov 20, 75(4), 387 - 92 The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products; Chamsart S et al.; Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy . Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied . Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer . Comparison has been made with unstressed material subjected to similar holding times . These tests essentially show that under all these conditions, <2% chDNA was present in the pDNA solution, the pDNA itself was not fragmented, and a yield of 1 mg/g cell was obtained . These results, together with studies of rheological properties, have led to the design of a 60-L, stirred lysis reactor and the production of high-quality pDNA solution with <1% chDNA after further purification . Semin Thromb Hemost, 2001 Oct, 27(5), 437 - 43 Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide; Naggi A et al.; A process to generate glycosaminoglycans with heparin- and heparan sulfate-like sequences from the Escherichia coli K5 capsular polysaccharide is described . This polymer has the same structure as N-acetylheparosan, the precursor in heparin/ heparan sulfate biosynthesis . The process involves chemical N-deacetylation and N-sulfation, enzymatic conversion of up to 60% of the D-glucuronic acid to L-iduronic acid residues, and chemical O-sulfation . Because direct sulfation afforded unwanted 3-O-sulfated (instead of 2-O-sulfated) iduronic acid residues, a strategy involving graded solvolytic desulfation of chemically oversulfated C5-epimerized sulfaminoheparosans was assessed using persulfated heparin and heparan sulfate as model compounds . The O-desulfation process was shown to increase the anti-factor Xa activity of oversulfated heparin. Pharmacogenetics, 2001 Oct, 11(7), 609 - 17 Polymorphism of human Alpha class glutathione transferases; Tetlow N et al.; The recognition of the importance and utility of single nucleotide polymorphisms has generated an interest in the development of new strategies for their identification . Analysis of the Expressed Sequence Tag (EST) database can provide a rapid and efficient means of identifying polymorphisms . Screening of the Alpha class glutathione transferases (GSTs) in the EST database identified 10 putative polymorphisms in the coding region of the GSTA1 and GSTA2 genes, six of which were subsequently verified by sequence analysis . Polymerase chain reaction/restriction fragment length polymorphism analysis revealed the existence of three variants, a silent base substitution, K125K (G365A) in GSTA1, and T112S and E210A in GSTA2, in European Australian, African and Chinese populations . The variant isoforms of GSTA2 were expressed in Escherichia coli, purified, and enzymatically characterized . Modelling of the two GSTA2 polymorphisms into a three-dimensional structure of GSTA2, and characterization of their enzymatic properties, has shown that the structure and function of the wild-type GSTA2-2 isoenzyme is not significantly altered by these polymorphisms . This report demonstrates that analysis of the EST database provides a rapid and efficient means of identifying variant proteins. Pharmacogenetics, 2001 Oct, 11(7), 597 - 607 Polymorphisms in human CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid; Dai D et al.; Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (Taxol) . It is also the predominant P450 responsible for the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs) in human liver and kidney . In this study, we describe two new CYP2C8 alleles containing coding changes: CYP2C8*2 has an Ile269Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8 . CYP2C8*2 was found only in African-Americans, while CYP2C8*3 occurred primarily in Caucasians . Neither occurred in Asians . The frequency of the CYP2C8*2 allele was 0.18 in African-Americans, and that of CYP2C8*3 was 0.13 in Caucasians . CYP2C8*1 (wild-type), CYP2C8*2 and CYP2C8*3 cDNAs were expressed in Escherichia coli, and the ability of these enzymes to metabolize both paclitaxel and arachidonic acid was assessed . Recombinant CYP2C8*3 was defective in the metabolism of both substrates . The turnover number of CYP2C8*3 for paclitaxel was 15% of CYP2C8*1 . CYP2C8*2 had a two-fold higher Km and two-fold lower intrinsic clearance for paclitaxel than CYP2C8*1 . CYP2C8*3 was also markedly defective in the metabolism of arachidonic acid to 11,12- and 14,15-EET (turnover numbers 35-40% that of CYP2C8*1) . Thus, CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel and the physiologically important compound arachidonic acid . This polymorphism has important clinical and physiological implications in individuals homozygous for this allele. J Vet Med B Infect Dis Vet Public Health, 2001 Sep, 48(7), 501 - 12 Sows intramammarily inoculated with Escherichia coli influence of time of infection, hormone concentrations and leucocyte numbers on development of disease; Magnusson U et al.; The aim of this study was to identify factors that influence the development of disease in sows inoculated with Escherichia coli in the mammary gland . Ten cross-bred primiparous sows were intramammarily inoculated with living E . coli bacteria at different time points before parturition: seven sows within 48 h before parturition and three sows approximately 96 h before parturition . Before and after inoculation, blood samples and mammary gland biopsy specimens were collected and clinical observations were made . All seven sows inoculated close to parturition developed a rectal temperature of >39.5 degrees C during the first 48 h post-partum and two of them also showed other signs of clinical disease . In the sows inoculated 4 days before parturition, the rectal temperature never exceeded 39.5 degrees C during the first 48 h post-partum and none of them showed any other sign of clinical discase . There was a tendency (P < 0.1) that histological signs of mastitis were more frequent in the sows inoculated close to parturition . There were no overall differences between the two groups of sows in plasma concentrations of cortisol, oestradiol-17beta and 15-ketodihydro-PGF2alpha before inoculation . Before inoculation, the number of neutrophils in the blood was overall higher (P < 0.05) in the group of sows that were inoculated close to parturition . In comparison, the number of lymphocytes before inoculation had a tendency (P < 0.1) to be lower in that group . The data suggest that the time of infection of the mammary gland relative to parturition and the number of circulating neutrophils at the time of infection influence the development of chinical coliform mastitis in the sow. Appl Biochem Biotechnol, 2001 Jul, 95(1), 23 - 30 Construction, expression, and characterization of recombinant hirudin in Escherichia coli; Bi Q et al.; The mutant gene of HV2-K47 was obtained by polymerase chain reaction-directed mutagenesis and expressed in Escherichia coli . Many elements that could affect its expression level were compared . The product was purified to homogeneity via three chromatographic steps--ion exchange, gel filtration, and reverse phase chromatography--on the AKTA Explorer System . The anti-thrombin activity of HV2-K47 is much higher than that of recombinant HV2 . Some properties and expression conditions were investigated systematically, which would be useful for further studies of hirudin and other small proteins. Methods Enzymol, 2002, 345, 127 - 40 Expression, purification, and assay of cytosolic (catalytic) domains of membrane-bound mammalian adenylyl cyclases; Hatley ME et al.; The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes . They have permitted detailed mechanistic investigation of the actions of forskolin, Gs alpha, and the inhibitory G protein, Gi alpha . They have served as critical reagents for the development of plausible models of the catalytic mechanism of the enzyme . They have enabled X-ray crystallographic analysis of adenylyl cyclase; this technique was not approachable with the small quantities of the membrane-bound enzyme available previously . The information obtained by using the soluble domains of adenylyl cyclase has provided templates for description of the behavior of many forms of purine nucleotide cyclases from a variety of species . We now appreciate both adenylyl cyclases and guanylyl cyclases as dimeric enzymes with a 2-fold symmetrical domain arrangement (or pseudosymmetrical in the case of heterodimerization) . The active sites are located at the interface between the two domains, both of which contribute binding surfaces. Biol Pharm Bull, 2001 Oct, 24(10), 1102 - 7 Thermostability of doubly glycosylated recombinant lysozyme; Hashimoto Y et al.; We prepared a lysozyme mutant (Q41S/R61S) introducing Asn-type glycosylation signal sites by yeast expression system . On purification by cation exchange column at pH 7, three fractions were obtained . Peptide mapping and mass-spectrometry showed the fractions were the derivatives glycosylated at both Asn39 and Asn59, at only Asn39, and not glycosylated . It was revealed that the processing of Asn-linked oligosaccharide at Asn39 and Asn59 occurred independently in yeast cells . The denaturation temperatures of these derivatives by differential scanning calorimetry were 76.0, 68.8, and 67.5 degrees C at pH 3, respectively . The stabilization of glycosylated lysozyme depends on the degree of glycosylation . We concluded that stabilized proteins can be constructed by glycosylation at proper sites . Thermodynamic stabilization by the artificial double glycosylations on a protein has not yet been reported. Genetika, 2001 Aug, 37(8), 1055 - 62 {Features of functioning of the promoter of microcin C51 promoter under various conditions of Escherichia coli cell growth}; Veselovskii AM et al.; The level of transcription from the promoter of the microcin C51 operon (Pmcc) depends on the growth phase of Escherichia coli cells: transcription proceeds with low efficiency at the exponential phase of growth and with higher efficiency when growth of cells is delayed during entry into the stationary phase . The functioning of Pmcc was studied in cells grown in different media by a single-copy construct, which contained the cloned promoter region of the microcin C51 operon and the promoterless lac operon . A decrease in the rate of cell growth caused by changes in the sole carbon source in minimal medium correlated with an increase in the level of transcription from the Pmcc promoter at the exponential phase of growth; the expression of Pmcc-lac during cell entry into the stationary phase was higher under unfavorable medium conditions . The use of composite rich media impaired this feature . The addition of l-leucine (100 micrograms/ml) to the medium decreased the expression of Pmcc-lac in wild-type cells carrying the delta lrp mutation . A further increase in leucine concentration and the presence of other amino acids in the medium enhanced transcription that started from Pmcc during cell entry into the stationary growth phase . The capacity of the Pmcc promoter and of the wild-type lacZ gene promoter was virtually the same upon IPTG induction . A mutation in the ompR gene did not markedly influence transcription started from Pmcc. Bioorg Khim, 2001 Sep-Oct, 27(5), 364 - 71 {The dependence of stability of the green fluorescent protein-obelin hybrids on the nature of their constituent modules and the structure of the amino acid linker}; Skosyrev VS et al.; Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotide sequences were constructed . The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP-obelin hybrid proteins . These proteins were purified to homogeneity and subjected to limited trypsinolysis . It was shown that the resistance of GFP-obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules . The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers . Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases . The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure. J Ind Microbiol Biotechnol, 2001 Aug, 27(2), 94 - 103 Multiple mutations at the active site of naphthalene dioxygenase affect regioselectivity and enantioselectivity; Yu CL et al.; The importance of five amino acids at the active site of the multicomponent naphthalene dioxygenase (NDO) system was determined by generating site-directed mutations in various combinations . The substrate specificities of the mutant enzymes were tested with the substrates indole, indoline, 2-nitrotoluene (2NT), naphthalene, biphenyl, and phenanthrene . Transformation of these substrates measured the ability of the mutant enzymes to catalyze dioxygenation, monooxygenation, and desaturation reactions . In addition, the position of oxidation and the enantiomeric composition of products were characterized . All enzymes with up to three amino acid substitutions were able to catalyze dioxygenation reactions . A subset of these enzymes could also catalyze the monooxygenation of 2NT and desaturation of indoline . Single amino acid substitutions at positions 352 and 206 had the most profound effects on product formation . Of the single mutations made, only changes at position 352 affected the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene, but in the presence of the F352I mutation, changes at positions 206 and 295 also affected enantioselectivity . Major shifts in regioselectivity with biphenyl and phenanthrene resulted with several of the singly, doubly, and triply mutated enzymes . A new product not formed by the wild-type enzyme, phenanthrene cis-9,10-dihydrodiol, was formed as a major product from phenanthrene by enzymes with two (A206I/F352I) or three amino acid substitutions (A206I/F352I/H295I) . The results indicate that a variety of amino acid substitutions are tolerated at the active site of NDO. J Biol Chem, 2002 Jan 4, 277(1), 586 - 92 Epub 2001 Oct 18. The activity of Arabidopsis glycosyltransferases toward salicylic acid, 4-hydroxybenzoic acid, and other benzoates; Lim EK et al.; Benzoates are a class of natural products containing compounds of industrial and strategic importance . In plants, the compounds exist in free form and as conjugates to a wide range of other metabolites such as glucose, which can be attached to the carboxyl group or to specific hydroxyl groups on the benzene ring . These glucosylation reactions have been studied for many years, but to date only one gene encoding a benzoate glucosyltransferase has been cloned . A phylogenetic analysis of sequences in the Arabidopsis genome revealed a large multigene family of putative glycosyltransferases containing a consensus sequence typically found in enzymes transferring glucose to small molecular weight compounds such as secondary metabolites . Ninety of these sequences have now been expressed as recombinant proteins in Escherichia coli, and their in vitro catalytic activities toward benzoates have been analyzed . The data show that only 14 proteins display activity toward 2-hydroxybenzoic acid, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid . Of these, only two enzymes are active toward 2-hydroxybenzoic acid, suggesting they are the Arabidopsis salicylic acid glucosyltransferases . All of the enzymes forming glucose esters with the metabolites were located in Group L of the phylogenetic tree, whereas those forming O-glucosides were dispersed among five different groups . Catalytic activities were observed toward glucosylation of the 2-, 3-, or 4-hydroxyl group on the ring . To further explore their regioselectivity, the 14 enzymes were analyzed against benzoic acid, 3-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, and 2,6-dihydroxybenzoic acid . The data showed that glycosylation of specific sites could be positively or negatively influenced by the presence of additional hydroxyl groups on the ring . This study provides new tools for biotransformation reactions in vitro and a basis for engineering benzoate metabolism in plants. J Biol Chem, 2002 Jan 4, 277(1), 593 - 601 Epub 2001 Oct 18. Functional properties of a monoclonal antibody inhibiting the hepatitis C virus RNA-dependent RNA polymerase; Moradpour D et al.; The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), has recently emerged as a promising target for antiviral intervention . Here, we describe the isolation, functional characterization, and molecular cloning of a monoclonal antibody (mAb) inhibiting the HCV RdRp . This mAb, designated 5B-12B7, binds with high affinity to a conformational epitope in the palm subdomain of the HCV RdRp and recognizes native NS5B expressed in the context of the entire HCV polyprotein or subgenomic replicons . Complete inhibition of RdRp activity in vitro was observed at equimolar concentrations of NS5B and mAb 5B-12B7, whereas RdRp activities of classical swine fever virus NS5B and poliovirus 3D polymerase were not affected . mAb 5B-12B7 selectively inhibited NTP binding to HCV NS5B, whereas binding of template RNA was unaffected, thus explaining the mechanism of action at the molecular level . The mAb 5B-12B7 heavy and light chain variable domains were cloned by reverse transcription-PCR, and a single chain Fv fragment was assembled for expression in Escherichia coli and in eukaryotic cells . The mAb 5B-12B7 single chain Fv fragment bound to NS5B both in vitro and in transfected human cell lines and therefore may be potentially useful for intracellular immunization against HCV . More important, detailed knowledge of the mAb 5B-12B7 contact sites on the enzyme may facilitate the development of small molecule RdRp inhibitors as novel antiviral agents. J Biol Chem, 2001 Dec 7, 276(49), 46225 - 9 Epub 2001 Oct 18. Asymmetric recognition of DNA local distortion . Structure-based functional studies of eukaryotic Msh2-Msh6; Drotschmann K et al.; Crystal structures of bacterial MutS homodimers bound to mismatched DNA reveal asymmetric interactions of the two subunits with DNA . A phenylalanine and glutamate of one subunit make mismatched base-specific interactions, and residues of both subunits contact the DNA backbone surrounding the mismatched base, but asymmetrically . A number of amino acids in MutS that contact the DNA are conserved in the eukaryotic Msh2-Msh6 heterodimer . We report here that yeast strains with amino acids substituted for residues inferred to interact with the DNA backbone or mismatched base have elevated spontaneous mutation rates consistent with defective mismatch repair . Purified Msh2-Msh6 with substitutions in the conserved Phe(337) and Glu(339) in Msh6 thought to stack or hydrogen bond, respectively, with the mismatched base do have reduced DNA binding affinity but normal ATPase activity . Moreover, wild-type Msh2-Msh6 binds with lower affinity to mismatches with thymine replaced by difluorotoluene, which lacks the ability to hydrogen bond . The results suggest that yeast Msh2-Msh6 interacts asymmetrically with the DNA through base-specific stacking and hydrogen bonding interactions and backbone contacts . The importance of these contacts decreases with increasing distance from the mismatch, implying that interactions at and near the mismatch are important for binding in a kinked DNA conformation. J Biotechnol, 2001 Dec 28, 92(2), 195 - 204 Development of three different gene cloning systems for genetic investigation of the new species Amycolatopsis japonicum MG417-CF17, the ethylenediaminedisuccinic acid producer; Stegmann E et al.; For the first time gene cloning systems have been developed for Amycolatopsis japonicum . Direct transformation, polyethyleneglycol (PEG) induced protoplast transformation and conjugal transfer was established for A . japonicum MG417-CF17, the ethylenediaminedisuccinic acid (EDDS) producer . The direct transformation procedure was modified to introduce DNA . The most important parameter for an efficient DNA uptake was the age of the culture . Using of mycelium from 36-h old cultures resulted in the highest transformation frequencies . Further, conditions for transformation of A . japonicum protoplasts were established . The efficiency of transformation depended mainly on the source of PEG and the components of the regeneration agar . The replicative plasmid pULVK2A carrying pA-rep and the apramycin resistance gene was transferred into the EDDS producer with a frequency of 0.38 colonies microg(-1) DNA by using the direct transformation procedure and with a frequency of 0.56 colonies microg(-1) DNA by using the PEG induced protoplast transformation . The plasmid was genetically stable, and could easily be reisolated from A . japonicum . We also demonstrated that conjugal transfer of the plasmid pSET152 from Escherichia coli ET12567 (pUB307) to Amycolatopsis spores is possible . The plasmid pSET152 integrated in the A . japonicum chromosome . A titre of 2.4 x 10(-4) exconjugants per recipient was obtained. J Biotechnol, 2001 Dec 28, 92(2), 179 - 86 Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747; Ragnitz K et al.; The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized . Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A . aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60% . Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant . All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields . Therefore, the enzyme was highly purified and used in immobilization experiments . The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q . The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide . Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B. J Biotechnol, 2001 Dec 28, 92(2), 133 - 58 Modeling of inducer exclusion and catabolite repression based on a PTS-dependent sucrose and non-PTS-dependent glycerol transport systems in Escherichia coli K-12 and its experimental verification; Wang J et al.; We used genetically engineered sucrose positive Escherichia coli K-12 derivatives as a model system for the modeling and experimental verification of regulatory processes in bacteria . These cells take up and metabolize sucrose by the phosphoenolpyruvate (PEP)-dependent sucrose phosphotransferase system (Scr-PTS) . Expression of the scr genes, which cluster in two different operons (scrYAB and scrK), is negatively controlled by the ScrR repressor . Additionally, expression of the scrYAB operon, but not of the scrK operon is positively controlled by the cAMP-CRP complex . Modeling of sucrose transport and metabolism through the Scr-system and of the scr gene expression has been performed using a modular and object-orientated new approach . To verify the model and identify important model parameters we measured in a first set of experiments induction kinetics of the scr genes after growth on glycerol using strains with single copy lacZ operon fusions in the scrK or scrY genes, respectively . In a second set of experiments an additional copy of the complete scr-regulon was integrated into the chromosome to construct diplogenotic strains . Differences were observed in the induction kinetics of the cAMP-CRP-dependent scrY operon compared to the cAMP-CRP independent scrK operon as well as between the single copy and the corresponding diplogenotic strains. Proc Natl Acad Sci U S A, 1991 Jun 1, 88(11), 5046 - 50 Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold; Stallings WC et al.; 5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate . The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques . The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution . The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet . Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices . The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal . A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described . The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments . The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations. Proc Natl Acad Sci U S A, 1990 Feb, 87(4), 1446 - 50 Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants; Hanley-Bowdoin L et al.; The A component of the bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) encodes the viral protein (AL1) that is required for viral DNA replication . We have constructed transgenic Nicotiana benthamiana plants in which the AL1 open reading frame is transcribed under the control of the cauliflower mosaic virus 35S promoter . The transgenic plants, which were phenotypically normal, produced a single transcript from the 35S-AL1 construct and a 40-kDa protein that cross-reacted with a polyclonal antiserum raised against AL1 protein overproduced in Escherichia coli . Six of nine transgenic lines complemented a TGMV A variant with a mutation in AL1 when coinoculated with the B component of the TGMV genome . Single- and double-stranded forms of the B component were synthesized in leaf discs from a complementing, transgenic line in the absence of TGMV A . These results establish that the transgenic plants express functional AL1 protein and show that this viral protein is not only required, but sufficient, for single- and double-stranded replication of TGMV DNA in the presence of host proteins . These results also show that the AL1 protein is not by itself a determinant of disease or pathogenesis. J Biol Chem, 2001 Dec 28, 276(52), 49371 - 7 Epub 2001 Oct 17. The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity; Poplawski A et al.; The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication . The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme . The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type . In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli . The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme . The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity . These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif. Eur J Biochem, 2001 Oct, 268(20), 5430 - 8 Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans; Laurent V et al.; Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) cleaves the N-terminal X-Pro bond of peptides and occurs in mammals as both cytosolic and plasma membrane forms, encoded by separate genes . In mammals, the plasma membrane AP-P can function as a kininase, but little is known about the physiological role of the cytosolic enzyme . The C . elegans genome contains a single gene encoding AP-P (W03G9.4), analysis of which predicts regions displaying high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of key catalytic residues . The sequence of an EST (yk91g4), comprising the open reading frame of W03G9.4, confirmed the predicted genomic structure of the gene and the prediction that W03G9.4 codes for a nonsecreted protein with a molecular mass of 68 kDa . Nematodes transformed with a promoter reporter construct, W03G9.4:GFP, showed high levels of fluorescence in the intestine of larvae and adult hermaphrodites, indicating that the intestine is a major site of W03G9.4 expression . yk91g4 tagged with a hexahistidine and DLYDDDDK peptide epitope was expressed in Escherichia coli to yield, after affinity purification, a recombinant protein with a molecular mass of 71 kDa . The recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (RPPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NRVYIHPFHL), des-Arg bradykinin and AF1 (KNEFIRFamide) . The activity towards bradykinin was inhibited by EDTA and 1, 10 phenanthroline, as expected for a metalloenzyme, and also by apstatin (IC50, 1 microM), a selective inhibitor of mammalian AP-P . A Km of 45 microM and an optimum pH of 7-8 was observed with bradykinin as the substrate . The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+, Mn2+ and Zn2+ all inhibiting the hydrolysis of bradykinin . We conclude that W03G9.4 codes for a cytoplasmic AP-P with very similar enzymatic properties to those of mammalian AP-P, and we suggest that the enzyme has a physiological role in the intracellular hydrolysis of proline-containing peptides absorbed from the lumen of the intestine. Eur J Biochem, 2001 Oct, 268(20), 5270 - 7 Liberation of the intramolecular interaction as the mechanism of heat-induced activation of HSP90 molecular chaperone; Tanaka E et al.; The molecular chaperone function of HSP90 is activated under heat-stress conditions . In the present study, we investigated the role of the interactions in the heat-induced activation of HSP90 molecular chaperone . The preceding paper demonstrated two domain-domain interactions of HtpG, an Escherichia coli homologue of mammalian HSP90, i.e . an intra-molecular interaction between the N-terminal and middle domains and an intermolecular one between the middle and C-terminal domains . A bacterial two-hybrid system revealed that the two interactions also existed in human HSP90alpha . Partners of the interaction between the N-terminal and middle domains of human HSP90alpha could, but those between the middle and C-terminal domains could not, be replaced by the domains of HtpG . Thus, the interface between the N-terminal and middle domains is essentially unvaried from bacterial to human members of the HSP90-family proteins . The citrate synthase-binding activity of HtpG at an elevated temperature was solely localized in the N-terminal domain, but HSP90alpha possessed two sites in the N-terminal and other domains . The citrate-synthase-binding activity of the N-terminal domain was suppressed by the association of the middle domain . The complex between the N-terminal and middle domains is labile at elevated temperatures, but the other is stable even at 70 degrees C . Taken together, we propose the liberation of the N-terminal client-binding domain from the middle suppressor domain is involved in the temperature-dependent activation mechanism of HSP90 molecular chaperone. Eur J Biochem, 2001 Oct, 268(20), 5258 - 69 Domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone; Nemoto TK et al.; In the present study, we investigated the domain structure and domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 . Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e . Arg7-Gly8, Arg336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90alpha . A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions . Thus, HtpG consists of three domains, i.e . Domain A, Met1-Arg336; domain B, Glu337-Lys552; and domain C, Leu553-Ser624, as does HSP90 . The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions . As a result, two kinds of domain-domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer . Domain B could be structurally and functionally divided into two subdomains, the N-terminal two-thirds (subdomain BI) that interacted with domain A and the C-terminal one-third (subdomain BII) that interacted with domain C . The C-terminal two-thirds of domain A, i.e . Asp116-Arg336, were sufficient for the binding to domain B . We finally propose the domain organization of an HtpG dimer. Res Microbiol, 2001 Sep, 152(7), 663 - 9 Amount and redox state of cytoplasmic, membrane and periplasmic proteins in Escherichia coli redox mutants; de Crouy-Chanel A et al.; We analyzed the amount and redox state of cytoplasmic, membrane and periplasmic proteins in Escherichia coli mutants deficient in thioredoxin, thioredoxin reductase, glutathione and Ds