Characterization of the proteins encoded by the<i>Bacillus subtilis yoxA-dacC</i>â operon

Duez, Colette; Zervosen, Astrid; Teller, Nathalie; Melkonian, Rémy; Banzubazé, Emmanuel; Bouillenne, Fabrice; Luxen, André; Frère, Jean‐Marie

doi:10.1111/j.1574-6968.2009.01761.x

Cited by 5 publications

(9 citation statements)

References 24 publications

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“…The much greater effectiveness of the extended nucleophiles mentioned above than D-alanine (Table 3) indicates that the R39 DD-peptidase is probably a more effective endopeptidase than carboxypeptidase. This conclusion agrees with most current views of the in vivo role of LMM class C (47). An endopeptidase substrate 15, which is not susceptible to the carboxypeptidase or transpeptidase (here defined as beginning by D-alanine displacement) reactions, has been described.…”

Section: Methodssupporting

confidence: 89%

Kinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates

et al. 2010

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The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

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Section: Methodssupporting

confidence: 89%

Kinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates

et al. 2010

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“…Another possible role for PBP4a that has been suggested is in biofilm formation. 66 In vivo measurements of the inhibitory power of boronates 2−4 against E. coli PBPs in intact cells were also made, with results very similar to those in the membranes. The HMM enzymes were not affected.…”

Section: ■ Results and Discussionmentioning

confidence: 85%

Inhibition of Bacterial DD-Peptidases (Penicillin-Binding Proteins) in Membranes and in Vivo by Peptidoglycan-Mimetic Boronic Acids

2012

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The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K i < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.

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“…It thus may be that BsPBP4a is, like the R39 DD-peptidase, an endopeptidase, 41 preferring a free N-terminus adjacent to the cleavage site. In solution, however, it may exist in a largely inactive conformation (hence the high K m values for 5 and 9) from which an active conformation can be induced only poorly by a specific substrate such as 5 or 9 or, more effectively, by a boronate transition-state analogue such as 11 (e.g., Scheme 2).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

et al. 2011

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The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and lowmolecular mass (LMM) enzymes. The latter group, which is subdivided into classes A−C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 DD-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown. T he bacterial DD-peptidases catalyze the final steps of peptidoglycan (cell wall) biosynthesis 1,2 and are efficiently inhibited by β-lactam antibiotics.3 Resistance to these antibiotics continues to emerge, particularly from evolution of new β-lactamases, enzymes that catalyze hydrolytic destruction of β-lactams, but also by the evolution and dispersal of β-lactam-resistant DD-peptidases. and Neisseria gonorrhoeae. 10Many attempts have been made to develop new β-lactams effective against mutant 11,12 but, to date, these have not been completely successful. The ideal of a broadspectrum β-lactam not susceptible to loss of effectiveness through DD-peptidase mutation has been difficult to achieve. To a considerable extent, this is likely due to the largely trial and error approach to new β-lactams. The alternative approach of targeting the reaction center and the common and essential structural features of the DD-peptidase active site has not been pursued as vigorously. C...

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Characterization of the proteins encoded by theBacillus subtilis yoxA-dacCâ operon

Cited by 5 publications

References 24 publications

Kinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates

Kinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates

Inhibition of Bacterial DD-Peptidases (Penicillin-Binding Proteins) in Membranes and in Vivo by Peptidoglycan-Mimetic Boronic Acids

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

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Characterization of the proteins encoded by theBacillus subtilis yoxA-dacCâ operon

Cited by 5 publications

References 24 publications

Kinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates

Kinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates

Inhibition of Bacterial DD-Peptidases (Penicillin-Binding Proteins) in Membranes and in Vivo by Peptidoglycan-Mimetic Boronic Acids

Substrate Specificity of Low-Molecular Mass Bacterial dd-Peptidases

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Characterization of the proteins encoded by theBacillus subtilis yoxA-dacCâ operon