The f8-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is... more The f8-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is inducible by 8-lactam com- pounds. This regulation has been shown to depend on the products of two open reading frames, ORFI (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
Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29... more Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29-kDa p-lactamase (cefuroximase) with an unusually high activity towards the B-lactamase-stable oximino-cephalosporins (e.g . cefuroxime and cefotaxime). Sequencing of the corresponding gene, cumA, showed that the derived CumA p-lactamase belonged to the molecular class A. The structural gene was under the direct control of gene cumR, which was transcribed backwards and whose initiation codon was 165 bp away from that of the P-lactamase gene. This resembled the arrangement of structural and regulator genes ampC and ampR of the 39-kDa molecular-class-C p-lactamase AmpC present in many enterobacteria. Moreover, cloned genes ampD and ampG for negative modulation and signal transduction of AmpC p-lactamase induction, respectively, were also able to restore constitutively CumA overproducing and non-inducible I? vulgaris mutants to the inducible, wildtype phenotype. The results indicate that controls of the induction phenomena are equivalent for the CumA and AmpC P-lactamase. Very different structural genes can thus be under the control of identical systems.
In the analysis of the interactions between ,-lactam antibiotics and their target enzymes, it is ... more In the analysis of the interactions between ,-lactam antibiotics and their target enzymes, it is often difficult to estimate the kinetic properties of the molecules which react rapidly with their targets and in consequence behave as the most efficient anti- biotics. The combined utilization of fluorescein-labelled penicillins and of a new competition method has allowed an accurate determination of the high second-order rate constants characterizing the acylation of Bacillus licheniformis penicillin-U.S.A.); f-iodopenicillanic acid (fl-IP) was from Pfizer Central Research (Sandwich, Kent, U.K.); benzylpenicillin was from Rhone-Poulenc (Paris, France); ceftazidime was from Glaxo Group Research (Greenford, Middx., U.K.); and cefotaxime was from Hoechst-Roussel (Romainville, France). All these compounds (for the structures see Matagne et al. [8]) were kindly given by the respective companies.
Journal of the American Chemical Society, Jun 24, 2011
Penicillin-binding proteins (PBPs) are DD-peptidases involved in the late stages of peptidoglycan... more Penicillin-binding proteins (PBPs) are DD-peptidases involved in the late stages of peptidoglycan synthesis. Their natural substrate is the C-terminal D-Ala-D-Ala dipeptide of the stem pentapeptide necessary to cross-link the glycan chains of the peptidoglycan. 3 A series of compounds, mainly containing variations on the theme of the β-lactam ring, have proven their efficacy as PBP inhibitors. For over 60 years, penicillins, cephalosporins, monobactams, and carbapenems have been used to combat all types of bacterial infections and continuously developed in response to the progressive bacterial resistance to β-lactam antibiotics. Recent research has intensified the investigation of non-β-lactam inhibitors of PBPs, e.g., lactivicin, a bicyclic molecule with a γ-lactam/ γ-lactone ring structure, 4,5 and shown that boronic acids represent potential inhibitors of PBPs. 6À8 It has been known for a long time that serine amidohydrolases are inhibited by boronic acids, and structures of complexes between boronic acids and trypsin, chymotrypsin, subtilisin, elastase, R-lytic protease, serine β-lactamases, prolyl tripeptidyl aminopeptidase, and hormone-processing protease have been described extensively. 9À16 Most structures with boronic acids show the boron atom in a tetrahedral conformation, forming a covalent adduct with the catalytic serine, occupying 50 the oxyanion hole, and underlining the analogy with the transi-51 tion state of the catalytic process. Escherichia coli PBP5 was the first PBP for which the crystallo-53 graphic structure of a boronic acid complex was described. This 54 structure has helped decipher the deacylation mechanism of 55 penicillin-binding proteins. More recently, we described the struc-56 ture of the DD-peptidase of Actinomadurax sp. R39 (R39) with an 57 ethylboronic acid bearing the side chain of diaminopimelic acid, a 58 characteristic amino acid of the peptidoglycan, providing a view of a 59 transition state close to the one that is expected with the natural 60 substrate of PBPs. 6 61 Although almost all of these structures reveal the boron 62 involved in a single dative covalent bond with the catalytic serine, 63 other covalent complexes are possible between serine proteases 64 and boronic acids. A dicovalent complex was first observed with 65 γ-chymotrypsin and D-p-chlorophenyl-or D-1-naphthylboronic 66 acids. 15 Both compounds linked to Ser195Oγ and His57Nε2, 67 two residues of the catalytic triad of γ-chymotrypsin. Likewise, 68 dicovalent complexes with borate and benzylboronate linked to
are highly conserved residues in class A fl-lactamases forming one wall of the active- site cavit... more are highly conserved residues in class A fl-lactamases forming one wall of the active- site cavity. All three residues of the SDN loop in Streptomyces albus G fl-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser'30 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser'30 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp'3' into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn'l32 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat being the most affected parameter, which would indicate a role of Asn'32 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described Nl 32S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.
Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylg... more 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.
The sequences of three Enterobacter cloacae ampC ,?-lactamase genes have been determined. The ded... more The sequences of three Enterobacter cloacae ampC ,?-lactamase genes have been determined. The deduced amino acid sequences are very similar: out of a total of 361 residues, only eight positions were found to be variable, and several mutations yielded residues with very similar properties. The kinetic properties of two of the enzymes were not significantly different. The three enzymes also exhibited a high degree of homology (> 70 %) with the ampC ,J-lactamases of Escherichia coli K12 and Citrobacter freundii, confirming the homogeneity of class-C ,8-lactamases. Abbreviations used: bp, base-pair(s); f.p.l.c., fast protein liquid chromatography. § To whom correspondence and reprint requests should be sent.
In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K... more In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wildtype (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.
The previously cloned class A /Mactamase gene (bla) of Streptomyees caeaoi was shown to be induci... more The previously cloned class A /Mactamase gene (bla) of Streptomyees caeaoi was shown to be inducible by /Mactam 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/%lactamase production. ORFI codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C /%lactamase) genes in several Enterobacteria.
Purpose: The PBP4* is a Penicillin Binding Protein belonging to the class C of AmpH type whose fu... more Purpose: The PBP4* is a Penicillin Binding Protein belonging to the class C of AmpH type whose function remains poorly understood. This study aimed to evaluate the biophysical and enzymatic properties of the Bacillus subtilis PBP4* to gain insights into its role in the context of bacterial cell wall recycling. Methods: To characterize the PBP4*, the full-length PBP4* and its N-terminal penicillin-binding domain have been produced in Escherichia coli and purified. Results: A comparison of biophysical properties has shown that both recombinant proteins are monomeric in solution and retain the same thermal stability. On the other hand, the D-alanine methyl esterase activity detected with the full-length PBP4* is impeded by the cleavage of the 92 amino acid C-terminal domain. The esterase activity of the full-length PBP4* demonstrates a clear D-stereospecificity. The PBP4* is also active on B. subtilis cell walls bearing teichoic acids, compounds commonly substituted with D-alanine residues. Conclusions: Our results are in agreement with the hypothesis that PBP4* could play a role in recycling cell wall components, as previously suggested.
In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K... more In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the cr...
Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-b... more Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-dir...
The f8-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is... more The f8-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is inducible by 8-lactam com- pounds. This regulation has been shown to depend on the products of two open reading frames, ORFI (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
Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29... more Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29-kDa p-lactamase (cefuroximase) with an unusually high activity towards the B-lactamase-stable oximino-cephalosporins (e.g . cefuroxime and cefotaxime). Sequencing of the corresponding gene, cumA, showed that the derived CumA p-lactamase belonged to the molecular class A. The structural gene was under the direct control of gene cumR, which was transcribed backwards and whose initiation codon was 165 bp away from that of the P-lactamase gene. This resembled the arrangement of structural and regulator genes ampC and ampR of the 39-kDa molecular-class-C p-lactamase AmpC present in many enterobacteria. Moreover, cloned genes ampD and ampG for negative modulation and signal transduction of AmpC p-lactamase induction, respectively, were also able to restore constitutively CumA overproducing and non-inducible I? vulgaris mutants to the inducible, wildtype phenotype. The results indicate that controls of the induction phenomena are equivalent for the CumA and AmpC P-lactamase. Very different structural genes can thus be under the control of identical systems.
In the analysis of the interactions between ,-lactam antibiotics and their target enzymes, it is ... more In the analysis of the interactions between ,-lactam antibiotics and their target enzymes, it is often difficult to estimate the kinetic properties of the molecules which react rapidly with their targets and in consequence behave as the most efficient anti- biotics. The combined utilization of fluorescein-labelled penicillins and of a new competition method has allowed an accurate determination of the high second-order rate constants characterizing the acylation of Bacillus licheniformis penicillin-U.S.A.); f-iodopenicillanic acid (fl-IP) was from Pfizer Central Research (Sandwich, Kent, U.K.); benzylpenicillin was from Rhone-Poulenc (Paris, France); ceftazidime was from Glaxo Group Research (Greenford, Middx., U.K.); and cefotaxime was from Hoechst-Roussel (Romainville, France). All these compounds (for the structures see Matagne et al. [8]) were kindly given by the respective companies.
Journal of the American Chemical Society, Jun 24, 2011
Penicillin-binding proteins (PBPs) are DD-peptidases involved in the late stages of peptidoglycan... more Penicillin-binding proteins (PBPs) are DD-peptidases involved in the late stages of peptidoglycan synthesis. Their natural substrate is the C-terminal D-Ala-D-Ala dipeptide of the stem pentapeptide necessary to cross-link the glycan chains of the peptidoglycan. 3 A series of compounds, mainly containing variations on the theme of the β-lactam ring, have proven their efficacy as PBP inhibitors. For over 60 years, penicillins, cephalosporins, monobactams, and carbapenems have been used to combat all types of bacterial infections and continuously developed in response to the progressive bacterial resistance to β-lactam antibiotics. Recent research has intensified the investigation of non-β-lactam inhibitors of PBPs, e.g., lactivicin, a bicyclic molecule with a γ-lactam/ γ-lactone ring structure, 4,5 and shown that boronic acids represent potential inhibitors of PBPs. 6À8 It has been known for a long time that serine amidohydrolases are inhibited by boronic acids, and structures of complexes between boronic acids and trypsin, chymotrypsin, subtilisin, elastase, R-lytic protease, serine β-lactamases, prolyl tripeptidyl aminopeptidase, and hormone-processing protease have been described extensively. 9À16 Most structures with boronic acids show the boron atom in a tetrahedral conformation, forming a covalent adduct with the catalytic serine, occupying 50 the oxyanion hole, and underlining the analogy with the transi-51 tion state of the catalytic process. Escherichia coli PBP5 was the first PBP for which the crystallo-53 graphic structure of a boronic acid complex was described. This 54 structure has helped decipher the deacylation mechanism of 55 penicillin-binding proteins. More recently, we described the struc-56 ture of the DD-peptidase of Actinomadurax sp. R39 (R39) with an 57 ethylboronic acid bearing the side chain of diaminopimelic acid, a 58 characteristic amino acid of the peptidoglycan, providing a view of a 59 transition state close to the one that is expected with the natural 60 substrate of PBPs. 6 61 Although almost all of these structures reveal the boron 62 involved in a single dative covalent bond with the catalytic serine, 63 other covalent complexes are possible between serine proteases 64 and boronic acids. A dicovalent complex was first observed with 65 γ-chymotrypsin and D-p-chlorophenyl-or D-1-naphthylboronic 66 acids. 15 Both compounds linked to Ser195Oγ and His57Nε2, 67 two residues of the catalytic triad of γ-chymotrypsin. Likewise, 68 dicovalent complexes with borate and benzylboronate linked to
are highly conserved residues in class A fl-lactamases forming one wall of the active- site cavit... more are highly conserved residues in class A fl-lactamases forming one wall of the active- site cavity. All three residues of the SDN loop in Streptomyces albus G fl-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser'30 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser'30 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp'3' into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn'l32 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat being the most affected parameter, which would indicate a role of Asn'32 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described Nl 32S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.
Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylg... more 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.
The sequences of three Enterobacter cloacae ampC ,?-lactamase genes have been determined. The ded... more The sequences of three Enterobacter cloacae ampC ,?-lactamase genes have been determined. The deduced amino acid sequences are very similar: out of a total of 361 residues, only eight positions were found to be variable, and several mutations yielded residues with very similar properties. The kinetic properties of two of the enzymes were not significantly different. The three enzymes also exhibited a high degree of homology (> 70 %) with the ampC ,J-lactamases of Escherichia coli K12 and Citrobacter freundii, confirming the homogeneity of class-C ,8-lactamases. Abbreviations used: bp, base-pair(s); f.p.l.c., fast protein liquid chromatography. § To whom correspondence and reprint requests should be sent.
In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K... more In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wildtype (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.
The previously cloned class A /Mactamase gene (bla) of Streptomyees caeaoi was shown to be induci... more The previously cloned class A /Mactamase gene (bla) of Streptomyees caeaoi was shown to be inducible by /Mactam 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/%lactamase production. ORFI codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C /%lactamase) genes in several Enterobacteria.
Purpose: The PBP4* is a Penicillin Binding Protein belonging to the class C of AmpH type whose fu... more Purpose: The PBP4* is a Penicillin Binding Protein belonging to the class C of AmpH type whose function remains poorly understood. This study aimed to evaluate the biophysical and enzymatic properties of the Bacillus subtilis PBP4* to gain insights into its role in the context of bacterial cell wall recycling. Methods: To characterize the PBP4*, the full-length PBP4* and its N-terminal penicillin-binding domain have been produced in Escherichia coli and purified. Results: A comparison of biophysical properties has shown that both recombinant proteins are monomeric in solution and retain the same thermal stability. On the other hand, the D-alanine methyl esterase activity detected with the full-length PBP4* is impeded by the cleavage of the 92 amino acid C-terminal domain. The esterase activity of the full-length PBP4* demonstrates a clear D-stereospecificity. The PBP4* is also active on B. subtilis cell walls bearing teichoic acids, compounds commonly substituted with D-alanine residues. Conclusions: Our results are in agreement with the hypothesis that PBP4* could play a role in recycling cell wall components, as previously suggested.
In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K... more In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the cr...
Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-b... more Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-dir...
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Papers by Bernard Joris