Skalweit Marion J, Li Mei, Taracila Magda A
Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Antimicrob Agents Chemother. 2015 Mar;59(3):1472-7. doi: 10.1128/AAC.03537-14. Epub 2014 Dec 22.
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.
C类头孢菌素酶构成的威胁日益增大，而目前尚无这类酶的抑制剂。对大肠杆菌AmpC、P99和CMY-2酶中YXN环天冬酰胺的研究表明，青霉素或头孢菌素上C6′或C7′取代基与该天冬酰胺之间的相互作用对于确定底物特异性和酶稳定性很重要。我们试图对鲍曼不动杆菌临床上重要的ADC-7 C类β-内酰胺酶中的YXN环天冬酰胺进行特性分析。ADC-7中N148位的诱变产生了保留头孢菌素酶活性的功能性突变体（N152G、-S、-T、-Q、-A和-C）。使用标准测定法，我们发现N148G、-S和-T变体对头孢西丁和头孢洛林具有良好的催化活性，但头孢吡肟是一种较差的底物。由于另一类C类β-内酰胺酶CMY-2的N152变体因失活速率较高而更易被他唑巴坦抑制，我们还测试了ADC-7中的N148取代是否会影响砜类抑制剂、舒巴坦和他唑巴坦、A类β-内酰胺酶以及对ADC-7具有体外活性的鲍曼不动杆菌青霉素结合蛋白（PBP）抑制剂的失活作用。他唑巴坦和舒巴坦的50%抑制浓度（IC50）有所改善，N148S变体的IC50分别降低了7倍和2倍。N148S ADC-7酶与他唑巴坦形成的米氏复合物的同源模型显示，N148与抑制剂的砜部分之间的相互作用丧失。我们推测，这可能导致抑制剂的二级环更快速打开，因为未结合的砜是一个优良的离去基团，从而导致更快速地形成稳定的线性化抑制剂。
