Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein

Abstract

The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the β-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 β-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·S-1). Against a set of five α- and ε-substituted L-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for Nε-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an Nε-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 k2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed 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 Ac2-L-LyS-D-Ala-D- Ala (kcat/Km) was bell-shaped, with pKa 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.