Each of these groups can be further subdivided into three classes, A, B, and C, based on sequence analysis ( 3, 10). PBPs have molecular masses of 20-120 kDa and can be broadly divided into two groups, the low molecular mass (LMM) PBPs and the high molecular mass (HMM) PBPs. coli has eight classically known PBPs, labeled 1A, 1B, and 2-7, as well as several recent additions including PBP1C ( 7) and PBP6B ( 8) (recently reviewed in ( 9)). Cross-linked peptides are also cleaved by PBPs (endopeptidase activity, left to right up diagonal arrow).Įvery bacterial species has multiple PBPs, which are generally labeled in order of decreasing molecular mass. The acyl group can either be hydrolyzed (+H 2O) (hydrolase/ dd-carboxypeptidase activity) or transferred to the acceptor amino group on an adjacent cell wall peptide (right to left down diagonal arrow) to form a cross-link (transpeptidase activity). PBPs attack the cell wall peptide to release d-Ala and form an acyl-enzyme intermediate (the acyl group donor). For example, Streptomycs R61 has a Gly interpeptide bridge attached to the N ε-amino group of l,l-DAP ( 47). In Gram-negative bacteria a cross-link is formed directly between the N ε of the acyl group acceptor and the d-Ala carboxyl group of the acyl group donor, whereas in many Gram-positive bacteria a bridging amino acid or short peptide (interpeptide bridge) is found. Variations in the peptide substrate, such as substitution of l-Lys and other diamine acids in place of m-DAP, are often observed in Gram-positive bacteria (reviewed in ( 43)). Cell wall peptides are attached to a repeating NAG-NAM polysaccharide.
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The implications of this substrate specificity of NG PBP3 with respect to its possible role in cell wall biosynthesis, and for understanding the substrate specificity of the LMM PBPs in general, are discussed.Ĭell wall biosynthesis reactions catalyzed by the PBPs in most Gram-negative bacteria, including Neisseria gonorrhoeae ( 41, 42) and Actinomadura R39 ( 46). NG PBP3 demonstrated low selectivity for γ-D-Glu vs D-IsoGln, and for the presence or absence of the terminal L-Ala residue. This observation suggests that NG PBP3 is specific for the ∼D-Ala-D-Ala moiety of pentapeptides engaged in cross-links in the bacterial cell wall, such that NG PBP3 would act after transpeptidase-catalyzed reactions generate the acylated amino group required for its specificity. NG PBP3 demonstrated good catalytic efficiency (2.5 × 10 5 M −1sec −1) with the best of these substrates, with a pronounced preference (50-fold) for N ε-acylated substrates over N ε-nonacylated substrates. The capacity of these peptides to serve as substrates for Neisseria gonorrhoeae (NG) PBP3 was assessed.
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#Pbp3 neisseria series
A series of eight substrates, based on variation of the pentapeptide Boc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala, were synthesized to test specificity for three features of PBP substrates: 1) the presence or absence of an N ε-acyl group, 2) the presence of D-IsoGln in place of γ-D-Glu, and 3) the presence or absence of the N-terminal L-Ala residue. Despite their importance, their roles in cell wall biosynthesis remain enigmatic. Penicillin-binding proteins (PBPs) are bacterial enzymes involved in the final stages of cell wall biosynthesis, and are the lethal targets of β-lactam antibiotics.