Effect of asparagine substitutions in the YXN loop of a class C β-lactamase of Acinetobacter baumannii on substrate and inhibitor kinetics

Abstract

Class C cephalosporinases are a growing threat, and inhibitors of these enzymes are currently unavailable. Studies exploring the YXN loop asparagine in the Escherichia coli AmpC, P99, and CMY-2 enzymes have suggested that interactions between C6' or C7' substituents on penicillins or cephalosporins and this Asn are important in determining substrate specificity and enzymatic stability. We sought to characterize the YXN loop asparagine in the clinically important ADC-7 class C β-lactamase of Acinetobacter baumannii. Mutagenesis at the N148 position in ADC-7 yields functional mutants (N152G, -S, -T, -Q, -A, and -C) that retain cephalosporinase activity. Using standard assays, we show that N148G, -S, and -T variants possess good catalytic activity toward cefoxitin and ceftaroline but that cefepime is a poor substrate. Because N152 variants of CMY-2, another class C β-lactamase, are more readily inhibited by tazobactam due to higher rates of inactivation, we also tested if the N148 substitutions in ADC-7 would affect inactivation by sulfone inhibitors, sulbactam and tazobactam, class A β-lactamase, and A. baumannii penicillin-binding protein (PBP) inhibitors with in vitro activity against ADC-7. The 50% inhibitory concentrations (IC50s) for tazobactam and sulbactam were improved, with 7-fold and 2-fold reductions, respectively, for the N148S variant. A homology model of the N148S ADC-7 enzyme in a Michaelis-Menten complex with tazobactam showed a loss of interaction between N148 and the sulfone moiety of the inhibitor. We postulate that this may result in more-rapid secondary ring opening of the inhibitor, as the unbound sulfone is an excellent leaving group, leading to more-rapid formation of the stable linearized inhibitor.

FIG 1

Inhibitors with class C activity and ceftaroline fosamil.

FIG 2

(a) Michaelis-Menten complex of ADC-7 and tazobactam. (b) Michaelis-Menten complex of N148S ADC-7 variant and tazobactam.