Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin‐binding protein 4

Abstract

The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the b-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for b-lactam binding, DD-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30 000 M )1 AEs )1 ). Against a set of five a-and e-substituted L-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N e -acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N e -Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k 2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active sitedirected agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac 2 -L-Lys-D-Ala-D-Ala (k cat /K m ) was bell-shaped, with pK a values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other DD-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs.

Neisseria gonorrhoeae Virulence Factor NG1686 Is a Bifunctional M23B Family Metallopeptidase That Influences Resistance to Hydrogen Peroxide and Colony Morphology

“…Therefore, although NG1686 and HdpA may share dual activities, the residues responsible for activity differ between the proteins. The two other known gonococcal PG endopeptidases, PBP3 and PBP4, also have both endopeptidase and DD-carboxypeptidase activity, although they are not members of the M23B family (43,44).…”

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

“…Endopeptidation was not tested. One of the two LMM PBPs (PBP3 or PBP4) can be deleted without severely affecting the cells but the removal of both of them leads to a modest decrease of cell growth and a change in morphology, suggesting that N. gonorrhoeae PBP3 and PBP4 have a redundant function in normal cell wall biosynthesis (Stefanova et al , 2004).…”

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins)

“…Little evidence of substrate specificity has been observed and significantly enhanced rates were not observed with 14, 18 or more elaborate peptidoglycan-mimetic peptides [55,78]. Another ortholog, N. gonorrhoeae PBP4, exhibited similar low and non-specific activity against small peptides [55,79]. The Streptomyces K15 DD-peptidase is a membraneassociated, although not by means of a C-terminal peptide, LMMA enzyme [28,29].…”

“…pH rate profiles for several DD-peptidases have been determined, for poor and nonspecific substrates in most cases. A number, for example those of the Streptomyces R61 DD-peptidase (LMMB) [151], N. gonorrhoeae PBP3 (LMMC) [76], N. gonorrhoeae PBP4 (LMMA) [79], Actinomadura R39 DD-peptidase (LMMC) [S. A. Adediran and R. F. Pratt, unpublished data], and S. pneumoniae PBP2x (HMMB) [148], display typical bell-shaped curves for kcat/Km with pKas of 5 -7 and 7.5 -10. As usually interpreted, these results suggest a general base catalyst of pKa 5 -7.…”