Search

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.

The 22076-Mr Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak beta-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+/serine-containing beta-lactamases.

SummaryTo study the properties of the BlaR penicillin­receptor involved in the induction of the Bacillus licheniformis β­lactamase, the water­soluble carboxy terminal domain of the protein (BlaR­CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various ­lactamβ antibiotics were studied. The second­order acylation rate constants K 2 /K' ranged from 0.0017 to more than 1 µM ­1 s ­1 and the deacylation rate constants were lower than 4 x 10 ­5 s ­1 . These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR­CTD is thus one of the most sensitive penicillin­binding proteins presently described. In the light of these results, the kinetics of ­lactamase induction in β Bacillus licheniformis were re­examined. When starting with a rather high cell density, a good ­lactamase substrate such asβ benzylpenicillin is too sensitive to ­lactamase­mediated hydrolysis to allow full induction. By contrast, a poorβ β β­lactamase substrate (7­aminocephalosporanic acid) can fully derepress ­lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction.IntroductionThe

In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 microm by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the beta-lactamase locus (blaP-blaI-blaR1) were lower than (1.9 microm) or in the same range as (75 microm) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K(d)(OP1) = 1.7 +/- 0.5 10(-15) m(2), K(d)(OP2) = 3.3 +/- 0.9 10(-15) m(2), and K(d)(OP3) = 10.5 +/- 2.5 10(-15) m(2). The role of the DNA binding properties of BlaI on the beta-lactamase regulation is discussed.

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of b-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L. q 2003 Published by Elsevier Science Ltd

[en] Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels infuture transportation and energy production. However, current industrial hydrogen production processes, such assteam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, inparticular the use of fermentative microorganisms, have attracted scientific interest in recent years. However thelow overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailedunderstanding of the relationships between genome content, gene expression patterns, pathway utilisation andmetabolite synthesis is required to optimise the yield of biohydrogen production pathways.Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridiumbutyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when themetabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitoredin 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differenceswere observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and theywere pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated withconcurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation ofthe [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expressionlevels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, whilethe nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmentalfactors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/orthe nitrogenase.Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides amultidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism ofclostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways basedon genetic engineering techniques., info:eu-repo/semantics/published

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal cotyledons and characterized biochemically. The isolation procedure included acid and ammonium sulfate precipitations and anion and cation exchange chromatographies. The isolated PAGs have different NH2-terminal amino acid sequences (RGSXLTILPLRNMRDIVY, ISRVSXLTIHPLRNIMDML, and RGSNLTIHPLRNIRD) and apparent molecular masses (55, 57, and 59 kDa). Each shows several isoforms with different pI values. The three proteins share high sequence identity with each other and with other ovine, bovine, and caprine PAGs. They have not been described previously. The ovPAG-59 sequence differs from the previously identified ovPAG-4 sequence (determined by DNA cloning and sequencing) at only one position among the 15 N-terminal residues. The newly characterized ovPAGs and the procedure used to isolate them will be helpful in producing new antisera for investigating PAG secretion in pregnant ewes. Mol. Reprod. Dev. 64: 199–206, 2003. © 2003 Wiley-Liss, Inc.

Summary The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP β-lactamases by β-lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaR1 genes share a high degree of sequence homology and act as repressors and penicillin-sensory transducers respectively. It has been shown in S. aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study, we followed the fate of BlaI during β-lactamase induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blaI and blaR1 genes. In contrast to the situation in B. licheniformis 749/I, β-lactamase induction in B. subtilis 168/pDML995 was not correlated with the proteolysis of BlaI. To exclude molecular variations undetectable by SDS–PAGE, two-dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells. No variation in the BlaI isoelectric point was observed in induced cells, whereas the DNA-binding property was lost. Cross-linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non-covalent modification by a co-activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co-activator, our results show that the presence of penicillin stress is also required for full induction of β-lactamase biosynthesis.

The three diastereoisomers-(R,R), (S,S) and meso-of lanthionine were synthesized in aqueous solution with high diastereoselectivity (>99%). The (S) and (R) enantiomers of two differently protected sulfamidates were opened by nucleophilic attack of (R) or (S)-cysteine. Acidification and controlled heating liberated the free lanthionines. Using the same chemistry, an α-benzyl lanthionine was also prepared. The proposed method, which avoids the need of enrichment by recrystallization, opens the way to the labelling of these compounds with (35)S. Furthermore, in vivo bioincorporation into Escherichia coli W7 was studied. No incorporation of α-benzyl lanthionine was observed. In contrast, meso-lanthionine can effectively replace meso-diaminopimelic acid in vivo, while in the presence of (R,R)-lanthionine the initial increase of bacterial growth was followed by cell lysis. In the future, meso-[(35)S]lanthionine could be used to study the biosynthesis of peptidoglycan and its turnover in relation to cell growth and division.

Background: The l-aminopeptidase d-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal l and/or d-Ala residues from peptide substrates. This is the only known enzyme to liberate N-terminal amino acids with both d and l stereospecificity. The DmpA active form is an αβ heterodimer, which results from a putative autocatalytic cleavage of an inactive precursor polypeptide. Results: The crystal structure of the enzyme has been determined to 1.82 A resolution using the multiple isomorphous replacement method. The heterodimer folds into a single domain organised as an αββα sandwich in which two mixed β sheets are flanked on both sides by two α helices. Conclusions: DmpA shows no similarity to other known aminopeptidases in either fold or catalytic mechanism, and thus represents the first example of a novel family of aminopeptidases. The protein fold of DmpA does, however, show structural homology to members of the N-terminal nucleophile (Ntn) hydrolase superfamily. DmpA presents functionally equivalent residues in the catalytic centre when compared with other Ntn hydrolases, and is therefore likely to use the same catalytic mechanism. In spite of this homology, the direction and connectivity of the secondary structure elements differ significantly from the consensus Ntn hydrolase topology. The DmpA structure thus characterises a new subfamily, but supports the common catalytic mechanism for these enzymes suggesting an evolutionary relationship.

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (

The DmpA (D-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and D-alanine more efficiently than that of L-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an L configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is L-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (α-subunit) and 250-375 (β-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the β-subunit is followed by a hydrophobic peptide, which is predicted to form a β-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172.

beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics. A large number of enzymes have been described with very diverse primary structures and catalytic profiles. Nevertheless, all known three-dimensional structures of active-site serine beta-lactamases exhibit a high degree of similarity with apparently equivalent chemical functionalities in the same strategic positions. These groups might not, however, play identical roles in the various classes of enzymes. Structural data have also been recently obtained for the zinc metallo-beta-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied. Similarly, the induction of the synthesis of beta-lactamases is now better understood, but many questions remain to be answered.

The Bacillus subtilis strain 168 chromosomal region extending from 109† to 112° has been sequenced. Among the 35 ORFs identified, cotT and rapA were the only genes that had been previously mapped and sequenced. Out of ten ORFs belonging to a single putative transcription unit, seven are probably involved in hexuronate catabolism. Their sequences are homologous to Escherichia coli genes exuT, uidB, uxaA, uxaB, uxaC, uxuA and uxuB, which are all required for the uptake of free D-glucuronate, D-galacturonate and β-glucuronide, and their transformation into glyceraldehyde 3-phosphate and pyruvate via 2-keto-3-deoxygluconate. The remaining three ORFs encode two dehydrogenases and a transcriptional regulator. The operon is preceded by a putative catabolite-responsive element (CRE), located between a hypothetical promoter and the RBS of the first gene. This element, the longest and the only so far described that is fully symmetrical, consists of a 26 bp palindrome matching the theoretical B. subtilis CRE sequence. The remaining predicted amino acid sequences that share homologies with other proteins comprise: a cytochrome P-450, a glycosyltransferase, an ATP-binding cassette transporter, a protein similar to the formate dehydrogenase α-subunit (FdhA), a protein similar to NADH dehydrogenases, and three homologues of polypeptides that have undefined functions.

The production of beta-lactamase in Streptomyces cacaoi, which contains two beta-lactamase-encoding genes, blaL and blaU, is inducible by beta-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a beta-lactamase-negative species. The blaU clone did not respond to the presence of beta-lactams, whereas the blaL clone appeared to be inducible in S. lividans. The latter clone contains two open reading frames, blaA and blaB, located just upstream of but transcribed divergently from blaL, which were shown to be required for the production as well as the induction of BlaL. The deduced BlaA protein belongs to the LysR family of transcription regulators. In order to examine the role of BlaA in regulation, we here report on over-expression of a GST-BlaA fusion protein in Escherichia coli and its use for antibody preparation. The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA intergenic region. The BlaA DNA binding-site was further restricted to a 30-bp sequence containing a T-N11-A motif, a characteristic of LysR-type promoters. Another T-N11-A motif upstream of the blaU gene was also shown to bind BlaA. The affinities of these two T-N11-A motifs in BlaA binding were comparable. A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confer on an S. lividans host the capacity to produce inducible beta-lactamase. It can thus be concluded that the S. cacaoi blaL and blaU genes are controlled by the same regulatory system.

The catalytic pathway of class A β-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G β-lactamases yielded enzymes forming stable acyl-enzymes with β-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Ω-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the “catalytic” water molecule in both the acylation and the deacylation processes.

The opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5. In clinical strains, mutations in PBP5 further reduce its acylation rate by β-lactams. Previous studies have reported that ceftaroline had poor inhibitory activity against β-lactam-resistant E. faecium strains. In this study, we show that ceftaroline exhibits killing activity against our laboratory-derived ampicillin-resistant E. faecium mutant that overproduces a wild-type PBP5 and that ceftaroline inactivates PBP5 much faster than benzylpenicillin and faster than ceftobiprole.

Penicillin-binding proteins are a well established, validated and still a very promising target for the design and development of new antibacterial agents. Based on our previous discovery of several noncovalent small-molecule inhibitor hits for resistant PBPs we decided to additionally explore the chemical space around these compounds. In order to clarify their structure-activity relationships for PBP inhibition two new series of compounds were synthesized, characterized and evaluated biochemically: the derivatives of anthranilic acid and naphthalene-sulfonamide derivatives. The target compounds were tested for their inhibitory activities on three different transpeptidases: PBP2a from methicillin-resistant Staphylococcus aureus (MRSA) strains, PBP5fm from Enterococcus faecium strains, and PBP1b from Streptococcus pneumoniae strains. The most promising results for both of these series of compounds were obtained against the PBP2a enzyme with the IC50 values in the micromolar range. Although these results do not represent a significant breakthrough in the field of noncovalent PBP inhibitors, they do provide useful structure-activity relationship data, and thus a more solid basis for the design of potent and noncovalent inhibitors of resistant PBPs.

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still–Gennari olefination starting from Garner’s aldehyde. The Michaelis–Arbuzov reaction was used to form the phosphorus–carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi.

Molecular Dynamics is a method of choice for membrane simulations and the rising of coarse-grained forcefields has opened the way to longer simulations with reduced calculations times. Here, we present an elastic network, SAHBNET (Surface Accessibility Hydrogen-Bonds elastic NETwork), that will maintain the structure of soluble or membrane proteins based on the hydrogen bonds present in the atomistic structure and the proximity between buried residues. This network is applied on the coarse-grained beads defined by the MARTINI model, and was designed to be more physics-based than a simple elastic network. The SAHBNET model is evaluated against atomistic simulations, and compared with ELNEDYN models. The SAHBNET is then used to simulate two membrane proteins inserted in complex lipid bilayers. These bilayers are formed by self-assembly and the use of a modified version of the GROMACS tool genbox (which is accessible through the gcgs.gembloux.ulg.ac.be website). The results show that SAHBNET keeps the structure close to the atomistic one and is successfully used for the simulation of membrane proteins.

A 37LRP/p40 polypeptide is of major interest because it is consistently up-regulated in cancer cells in correlation with their invasive and metastatic phenotype. Furthermore, this polypeptide presents intriguing multifunctional properties because it has been characterized as the precursor of the metastasis-associated 67-kD laminin receptor (67LR) and as a cytoplasmic ribosomal-associated protein. The isolation of the 37LRP/p40 gene is a prerequisite for identifying the molecular mechanisms responsible for the constant up-regulation of the 67LR expression in cancer cells. To date, the active 37LRP/p40 gene has never been identified in any species due to the existence of multiple pseudogenes in most vertebrates genomes. In this study, we report for the first time the gene structure and potential regulatory sequences of the 37 LRP/p40 gene. The chicken genome was selected to undergo this characterization because it is the only known vertebrate that bears a single 37 LRP/p40 gene copy. The 37 LRP/p40 active gene is composed of 7 exons and 6 introns and bears features characteristic of a ribosomal protein gene. It does not bear a classical TATA box and it exhibits several transcription initiation sites as demonstrated by RNase protection assay and primer extension. Analysis of potential regulatory regions suggests that gene expression is driven not only by the 5' genomic region but also by the 5' untranslated and intron 1 sequences. On the basis of gene structure and extensive protein evolutionary study, we found that the carboxyterminal domain of the protein is a conserved lock-and-key structure/function domain that could be involved in the biosynthesis of the higher-molecular-weight 67-kD laminin receptor in vertebrates, whereas the central core of the protein would be responsible for the ribosome associated function. The first identification of the active 37LRP/p40 gene presented in this study is a critical step toward the isolation of the corresponding human gene and the understanding of the molecular mechanisms involved in the up-regulation of its expression during tumor invasion and metastasis.

Cosmid B577, a member of the collection of ordered clones corresponding to the genome of Mycobacterium leprae, contains a gene, provisionally called pon1, that encodes an 821-amino-acid-residue high-molecular-mass class A penicillin-binding protein, provisionally called PBP1. With similar amino acid sequences and modular designs, M. leprae PBP1 is related to Escherichia coli PBP1a and PBP1b, bienzymatic proteins with transglycosylase and transpeptidase activities. When produced in E. coli, His tag-labelled derivatives of M. leprae PBP1 adopt the correct membrane topology, with the bulk of the polypeptide chain on the surface of the plasma membrane. They defy attempts at solubilization with all the detergents tested except cetyltrimethylammonium bromide. The solubilized PBP1 derivatives can be purified by affinity chromatography on Ni2+-nitrilotriacetic acid agarose. They have low affinities for the usual penicillins and cephalosporins.

The aim of this study was to develop a method to identify cows illegally treated with bovine somatotropin (BST). Using the Western ligand blotting technique, BST treatment was shown to cause a dramatic decrease of IGF‐BP‐2 in the serum of treated cows. Measurement of the insulin‐like growth factor‐binding protein‐2 (IGF‐BP‐2) concentration in cow plasma could thus be a good way to identify BST‐treated animals. In order to develop an immunoassay specific for bovine IGF‐BP‐2, polyclonal antibodies against synthetic peptides of bovine IGF‐BP‐2 were produced. Three peptides were chosen in the [150–190] region of the molecule, which display no identity with other IGF‐BPs in serum: [154–165], [171–184] and [163–176]. Two antisera were obtained which reacted specifically with the entire bovine IGF‐BP‐2 molecule, both of which came from rabbits immunized with the [163–176] peptide.

Four GUC triplets in the coding region of the mRNA of interleukin 6 (IL-6) were examined for their suitability to serve as a target for hammerhead ribozyme-mediated cleavage. This selection procedure was performed with the intention to down-regulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribozymes for cleavage of IL-6 mRNA should be directed at this location.

The dnaK operon of Streptomyces coelicolor A3(2) was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two of the most conserved regions in 30 different DnaK proteins (HSP70). The isolated insert-a BamHI 5.6-kb fragment-was sequenced and shown to contain three open-reading frames organized in an operon and coding for proteins analogous to DnaK, GrpE and DnaJ, successively.

The beta-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is inducible by beta-lactam compounds. This regulation has been shown to depend on the products of two open reading frames, ORF1 (blaA) and ORF2 (blaB) [Lenzini, Magdalena, Fraipont, Joris, Matagne and Dusart (1992) Mol. Gen. Genet. 235, 41-48]. BlaA belongs to the LysR family of transcription activators, whereas BlaB shares some features with the penicillin-recognizing proteins. BlaB has now been overexpressed in Escherichia coli, purified and used for antibody preparation. Immunoblotting of cell-fractionated materials from S. cacaoi showed that BlaB is attached to the internal face of the cytoplasmic membrane. It could not be released by high salt concentrations or EDTA, but only by protease treatment. Under the assay conditions, BlaB did not act as a penicillin-binding protein, a beta-lactamase, a D-amino-peptidase or a target in a phosphorylation step.

In response to the widespread use of β-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent β-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC50: 1.3 μM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC50 = 26 μM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC50 = 138 μM) and a class C PBP (R39 from Actinomadura sp., IC50 = 0.6 μM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species.

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

β-Lactam antibiotics have long been a treatment of choice for bacterial infections since they bind irreversibly to Penicillin-Binding Proteins (PBPs), enzymes that are vital for cell wall biosynthesis. Many pathogens express drug-insensitive PBPs rendering β-lactams ineffective, revealing a need for new types of PBP inhibitors active against resistant strains. We have identified alkyl boronic acids that are active against pathogens including methicillin-resistant S. aureus (MRSA). The crystal structures of PBP1b complexed to 11 different alkyl boronates demonstrate that in vivo efficacy correlates with the mode of inhibitor side chain binding. Staphylococcal membrane analyses reveal that the most potent alkyl boronate targets PBP1, an autolysis system regulator, and PBP2a, a low β-lactam affinity enzyme. This work demonstrates the potential of boronate-based PBP inhibitors for circumventing β-lactam resistance and opens avenues for the development of novel antibiotics that target Gram-positive pathogens.

In Bacillus licheniformis 749/I, BlaP β-lactamase is induced by the presence of a β-lactam antibiotic outside the cell. The first step in the induction mechanism is the detection of the antibiotic by the membrane-bound penicillin receptor BlaR1 that is composed of two functional domains: a carboxy-terminal domain exposed outside the cell, which acts as a penicillin sensor, and an amino-terminal domain anchored to the cytoplasmic membrane, which works as a transducer-transmitter. The acylation of BlaR1 sensor domain by the antibiotic generates an intramolecular signal that leads to the activation of the L3 cytoplasmic loop of the transmitter by a single-point cleavage. The exact mechanism of L3 activation and the nature of the secondary cytoplasmic signal launched by the activated transmitter remain unknown. However, these two events seem to be linked to the presence of a HEXXH zinc binding motif of neutral zinc metallopeptidases. By different experimental approaches, we demonstrated that the L3 loop binds zinc ion, belongs to Gluzincin metallopeptidase superfamily and is activated by self-proteolysis.

d -Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d -glutamic acid to the cytoplasmic peptidoglycan precursor UDP- N -acetylmuramoyl- l -alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene- N -sulfonyl- d -Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d -Glu. The compounds that contained either constrained d -Glu or related rigid d -Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors. The binding modes of the best inhibitors were examined with high-resolution NMR spectroscopy and X-ray crystallography. We have solved a new crystal structure of the complex of MurD with an inhibitor bearing a 4-aminocyclohexane-1,3-dicarboxyl moiety. These data provide an additional step towards the development of sulfonamide inhibitors with potential antibacterial activities.

The use of an artificial consortium composed of selected hydrogen-producing species, instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In order to monitor such a consortium composed of different Clostridium spp., new protocols were tested for two different assays, FISH and qPCR. New species-specific FISH probes and qPCR primer sets were developed and optimised for three strains: Clostridium butyricum, Clostridium felsineum and Clostridium pasteurianum, that were used in a consortium. Application of a fast two-step FISH protocol, with pre-treatment step at 90 degrees C for 5 min and a subsequent hybridisation step at higher temperature (55 degrees C) for 20 min resulted in a much shorter analytical time compared to the standard FISH procedure (46 degrees C for 2-3 h) and gave a high hybridisation performance. Moreover, to accurately quantify each microorganism by qPCR assay, two innovations were tested: the direct use of cell lysates (omitting the DNA extraction step) and the use of two alternative molecular markers, recA and gyrA. These markers are present in single copies in the genome, whereas there are multiple copies of the ribosomal operons. This resulted in the development of accurate, reliable and fast FISH and qPCR assays for routine monitoring of the dynamics of artificial hydrogen-producing microbial consortia. Moreover, both techniques can be easily adapted to new Clostridium strains. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was successively replaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams. However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts. The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme. Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme. The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied. The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase.

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

The recombinant plasmid pAL-A3 bears a (poly ManA) alginate lyase-encoding gene that originates from the marine bacterium ATCC 433367 (Brown et al., Appl. Environ. Microbiol. (1991) 57, 1870-1872). The alginate lyase produced by Escherichia coli TC4 harbouring pAL-A3 was purified to protein homogeneity and the corresponding gene sequenced, giving access to the first known primary structure of an alginate lyase. The 265-amino acid residue alginate lyase showed lytic activity on a Pseudomonas aeruginosa alginate isolated from a cystic fibrosis patient. Unexpectedly, the alginate lyase thus characterized differed from that isolated from the culture medium of the bacterium ATCC 433367 (Romeo and Preston, Biochemistry (1986) 25, 8385-8391).

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific ‘carbapenemase’. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column.

In the green alga Chlamydomonas reinhardtii , phosphate starvation results in the accumulation of specific mRNAs. A cDNA clone bank was prepared from poly(A) + RNA isolated from cells grown on phosphate-derived (-P) medium. From this bank numerous cDNA clones representing RNAs that increased in abundance during phosphate limitation were selected by a different screening protocol with cDNA probes corresponding to cells grown in the pressence (+P) or in the absence (−P) of phosphate. An additional differential hybridization experiment on dots with plasmids extracted from 44 clones led to the selection of 26 clones displaying a stronger autoradiographic signal with (−P) probe. These clones were distributed among 13 classes, representing 13 RNA species. One cDNA from each class was partially or fully sequenced and the nucleotide and amino acid deduced sequences were compared to sequences of protein and nucleic acid databases. Surprisingly, no phosphatase sequence was identified in our library. Four sequences, however, revealed a high degree of identity with known sequences corresponding to enolase, pyruvate formate-lyase and P1 and L31 ribosomal proteins.

Among the large variety of micro-organisms capable of fermentative hydrogen production, strict anaerobes such as members of the genus Clostridium are the most widely studied. They can produce hydrogen by a reversible reduction of protons accumulated during fermentation to dihydrogen, a reaction which is catalysed by hydrogenases. Sequenced genomes provide completely new insights into the diversity of clostridial hydrogenases. Building on previous reports, we found that [FeFe] hydrogenases are not a homogeneous group of enzymes, but exist in multiple forms with different modular structures and are especially abundant in members of the genus Clostridium. This unusual diversity seems to support the central role of hydrogenases in cell metabolism. In particular, the presence of multiple putative operons encoding multisubunit [FeFe] hydrogenases highlights the fact that hydrogen metabolism is very complex in this genus. In contrast with [FeFe] hydrogenases, their [NiFe] hydrogenase counterparts, widely represented in other bacteria and archaea, are found in only a few clostridial species. Surprisingly, a heteromultimeric Ech hydrogenase, known to be an energy-converting [NiFe] hydrogenase and previously described only in methanogenic archaea and some sulfur-reducing bacteria, was found to be encoded by the genomes of four cellulolytic strains: Clostridum cellulolyticum, Clostridum papyrosolvens, Clostridum thermocellum and Clostridum phytofermentans.

Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the MurNAc and the α-amino group of L-Ala where the peptide chain was at least a dipeptide (L-Ala-γ-D-Glu) amidated by benzylamine on the γ-carboxyl group of D-Glu. In the presence of a tripeptide chain (L-Ala-γ-D-Glu-L-Lys) or a tetrapeptide chain (L-Ala-γ-D-Glu-m-A(2)pm-D-Ala) higher hydrolysis rates were observed. We have also demonstrated that the presence of TNB on the e-amino group of L-Lys only has a small influence on the hydrolysis capacity of sAmiD.

The previously cloned class A beta-lactamase gene (bla) of Streptomyces cacaoi was shown to be inducible by beta-lactam compounds in the host organism S. lividans. A regulatory region of 2.75 kb was identified and the nucleotide sequence determined. It contained four open reading frames (ORFs) of which only two were complete and required for induction. ORF1-ORF2 exerted a positive regulatory effect on the expression of bla. Inactivation of ORF1 or of ORF2 resulted not only in the loss of induction, but also in a 30- to 60-fold decrease in the basal (non-induced) level of beta-lactamase production. ORF1 codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C beta-lactamase) genes in several Enterobacteria.

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

The gene encoding the Bacillus sphaericus gamma-D-glutamyl-L-diamino acid endopeptidase II, a cytoplasmic enzyme involved in cell sporulation [1], contains the information for a 271-amino acid protein devoid of a signal peptide. The endopeptidase lacks sequence relatedness with other proteins of known primary structure except that its C-terminal region has significant similarity with the C-terminal region of the 54-kDa P54 protein of Enterococcus faecium, of unknown function [2].

Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a β-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.

AmiD is the fifth identified N -acetylmuramoyl- l -alanine zinc amidase of Escherichia coli . This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N -acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli . We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro- N -acetylmuramic-acid- l -Ala-γ- d -Glu- l -Lys, and the holoenzyme in complex with the l -Ala-γ- d -Glu- l -Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.

High-level production (880 mg liter −1 ) and isolation of the anteiso-C 17 isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp.