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Membrane-bound DD-carboxypeptidases from Bacillus megaterium KM general properties, substrate specificity and sensitivity to penicillins, cephalosporins and peptide inhibitors of the activity at pH5.

ABSTRACT: 1. The membrane from Bacillus megaterium KM contained a dd-carboxypeptidase with optimum activity under the following conditions: pH5.2, bivalent cation, 3mm; ionic strength, 40mm; temperature, 35 degrees C. It was inactivated by treatment with p-chloromercuribenzoate but was fairly insensitive to 2-mercaptoethanol. 2. The enzyme was inhibited by penicillins and cephalosporins. The inhibition of this enzyme was partially reversed on dialysis but 0.2m-2-mercaptoethanol could neither prevent nor reverse the inhibition. 3. The enzyme was extremely sensitive to changes in the configuration and size of the side chain of the C-terminal dipeptide of the substrate. An aliphatic side chain of a well-defined length and polarity was required in the residue that precedes the C-terminal dipeptide. 4. The enzyme was inhibited by a wide range of analogues of the peptidic portion of the natural substrate.

SUBMITTER: Maurino T

PROVIDER: S-EPMC1168394 | biostudies-other | 1974 Nov

REPOSITORIES: biostudies-other

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Project description:During the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between two meso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (between d-Ala and meso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. The dd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used by ld-transpeptidases as substrates to form 3-3 cross-links. Therefore, dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology of dd-CPases in mycobacteria is relatively unexplored. In this study, we deleted two dd-CPase genes, msmeg_2433 and msmeg_2432, both individually and in combination, from Mycobacterium smegmatis mc2155. Though the single dd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant, viz, a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to ?-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of the dd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCE The glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. The dd-CPases generate tetrapeptides by acting on the pentapeptides, and ld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of two dd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, including Mycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology of dd-CPases might help us design antimycobacterial therapeutics in the future.

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Membrane-bound DD-carboxypeptidases from Bacillus megaterium KM general properties, substrate specificity and sensitivity to penicillins, cephalosporins and peptide inhibitors of the activity at pH5.

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