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 ␤-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 ␤-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 ␤-lactamase, and A. baumannii penicillin-binding protein (PBP) inhibitors with in vitro activity against ADC-7. The 50% inhibitory concentrations (IC 50 s) 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. C lass C ␤-lactamases such as ADC-7, found in Acinetobacter baumannii, confer resistance to a wide variety of ␤-lactam antibiotics, including narrow-spectrum and extended-spectrum cephalosporins and penicillins (1, 2). Point mutations and deletions in the omega-loop or helices H2 or H10 and near the C terminus of the AmpC ␤-lactamases cause an expanded-spectrum AmpC (ESAC) phenotype (2, 3). At least 65 unique types of the ADC enzymes (4), including enzymes such as ADC-7, ADC-8, 3,5,6), have been described. The latter two enzymes have an ESAC phenotype. The inhibition behavior of ADC enzymes has been explored for boronate inhibitors and carbapenems (7) and for 7-alkylidenecephalosporin sulfones (8). We previously described the inhibition behavior of CMY-2 variants at the N152 position, with the N152G, -S, and -T variants showing enhanced k inact values for tazobactam (9) and increased 50% inhibitory concentrations (IC 50 s) for avibactam (10) (Fig. 1). Although not yet studied in the CMY-2 or ADC enzymes, the complex of avibactam with the AmpC of Pseudomonas aeruginosa, PDC-3, was recently obtained, revealing the key interactions of this novel, first-in-class diazabicyclooctane inhibitor with the YXN loop asparagine 152 in a class C active site (11,12). A more detailed study of the inhibition ...

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