Peptidoglycan carboxypeptidase

In addition to transpeptidases, other penicillin-binding proteins (PBPs) function as transglycosylases and carboxypeptidases. All of the PBPs are involved with assembly, maintenance, or regulation of peptidoglycan cell wall synthesis. When (3-lactam antibiotics inactivate PBPs, the consequence to the bacterium is a structurally weakened cell wall, aberrant morphological form, cell lysis, and death. 

A cephalosporin derivative 257 with structural features of the peptidoglycan has been conceived as an inhibitor specific for DD-transpeptidases <2003JA16322>. The compound 257 has been synthesyzed in 13 steps and has been tested with recombinant PBPlb and PBP5 of E. coli, a DD-transpetidase and a DD-carboxypeptidase, respectively. It has been found that compound 257 is a time-dependent and irreversible inhibitor of PBPlb and does not interact with PBP5, neither as an inhibitor (reversible or irreversible) nor as a substrate. 

The interactions of 8-lactams with PBPs indicate that these compounds are structural analogues of R-D-alanyl-D-alanine, the natural substrate of peptidoglycan transpeptidases and D-alanine carboxypeptidases, where R is the remainder of the pentapeptide. The mechanisms of the transpeptidase and carboxypeptidase reactions are thought to involve formation of an acyl-enzyme intermediate that can react with either a primary amine (e.g., an a-amino group) to form a peptide bond, or with water to form a carboxylic acid. In both reactions D-alanine is released before the acyl enzyme is formed. When a S-lactam antibiotic enters the binding site, the /1-lactam bond is hydrolyzed, and the resulting acyl group reacts with the active-site... 

VanX is a carboxypeptidase that hydrolyzes the amide bond of a dipeptide, D-alanyl-D-alanine (D-Ala-D-Ala). This enzyme is essential for vancomycin-resistant enterococci (VRE), because it produces peptidoglycan precursors terminating in D-alanyl-D-lactate (D-Ala-D-Lac) in place of D-Ala-D-Ala, resulting in a 100-fold decrease in the affinity with vancomycin.Scheme 5 depicts a proposed mechanism of VanX, where the active-site structure is similar to those of CPDA and thermolysin. The Zn -bound H2O activated by Glul81 attacks... 

A carboxypeptidase-transpeptidase, which incorporates free diaminopimelic acid into previously formed cell walls, has been isolated from Bacillus mega-terium Membranes from B. megaterium have been solubilized and then reconstituted in a form capable of synthesizing peptidoglycan. Using this reconstituted system, an assay has been developed for a factor necessary for... 

The mechanism of peptidoglycan synthesis in Pseudomonas aeruginosa appears to be similar to that of Escherichia coli, although evidence has suggested that in the former the DD-carboxypeptidase and transpeptidase systems may have somewhat different specificity restrictions from those in E. colif Some... 

The situation in Gram-positive bacteria is more complex because of the wide variation in peptidoglycan structure (Ghuysen, 1977). The major PBP is a low molecular weight D,D-carboxypeptidase but in none has it been demonstrated unequivocally to be the lethal target. 

When bacteria are exposed to 3-lactams a small amount of the antibiotic becomes covalently linked to the cells. The amount bound varies with the nature of the organism and the antibiotic but it is always very low, of the order of 200-4000 molecules per cell. These covalently tound molecules are responsible for damaging and, ultimately, killing the cell. They are bound to transpeptidases and carboxypeptidases and effectively inhibit the enzymes. Since no other metabolic or biosynthetic activities are affected the cells continue to grow but are unable to produce cross-linked peptidoglycan. Abnormal cell shapes develop and the cells eventually stop growing. The precise fate of the cells depends upon the nature and the concentration of the 3"lactam involved and upon the organism itself. Lysis is a common phenomenon but filaments and cells with other unusual shapes can persist without lysis. 

The mechanism by which penicillin becomes covalently linked to the transpeptidases and carboxypeptidases is illustrated in Figure 1.17. The 3-lactam bond of the antibiotic is broken by the enzyme forming an inactive penicilloyl-enzyme intermediate. The intermediates are sufficiently stable to ensure that all of the transpeptidase and carboxypeptidase molecules in the cell are inactivated and cross-linking of the peptidoglycan cannot occur. Enzyme activity is only slowly restored as the penidlloyl-enzyme complexes break down yielding penicillin fragments. It may not be... 

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