Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4.

The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the beta-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 beta-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 alpha- and epsilon-substituted l-Lys-D-Ala-D-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N epsilon-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N epsilon-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.

Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities.

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.

Potential transition state analogue inhibitors for the penicillin-binding proteins.

Penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis. The development of new PBP inhibitors is a potentially viable strategy for developing new antibacterial agents. Several potential transition state analogue inhibitors for the PBPs were synthesized, including peptide chloromethyl ketones, trifluoromethyl ketones, aldehydes, and boronic acids. These agents were characterized chemically, stereochemically, and as inhibitors of a set of low molecular mass PBPs: Escherichia coli (EC) PBP 5, Neisseria gonorrhoeae (NG) PBP 3, and NG PBP 4. A peptide boronic acid was the most effective PBP inhibitor in the series, with a preference observed for a d-boroAla-based over an l-boroAla-based inhibitor, as expected given that physiological PBP substrates are based on d-Ala at the cleavage site. The lowest K(I) of 370 nM was obtained for NG PBP 3 inhibition by Boc-l-Lys(Cbz)-d-boroAla (10b). Competitive inhibition was observed for this enzyme-inhibitor pair, as expected for an active site-directed inhibitor. For the three PBPs included in this study, an inverse correlation was observed between the values for log K(I) with 10b and the values for log(k(cat)/K(m)) for activity against the analogous substrate, and K(m)/K(I) ratios were 90, 1900, and 9600 for NG PBP 4, EC PBP 5, and NG PBP 3, respectively. These results demonstrate that peptide boronic acids can be effective transition state analogue inhibitors for the PBPs and provide a basis for the use of these agents as probes of PBP structure, function, and mechanism, as well as a possible basis for the development of new PBP-targeted antibacterial agents.

pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5.

The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure-function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five alpha- and epsilon-substituted L-Lys-D-Ala-D-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6-12.3, and the k(cat)/K(m) vs. pH profile for activity against Ac(2)-L-Lys-D-Ala-D-Ala was bell-shaped, with pK(a)s at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed DD-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A beta-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.