Resistance to �-lactam antibiotics in the pathogenic bacteria, Acinetobacter baumannii, presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance derives from the expression of class C �-lactamase enzymes, known as Acinetobacter-Derived Cephalosporinases (ADCs). In the search for novel therapies for multidrug resistant pathogens, determining the molecular structure of the enzyme active site is an important contribution. Here, we have determined the X-ray crystal structure of the extended-spectrum class C �-lactamase, ADC-7, at 1.7 � (P1 space group). In addition, while current �-lactamase inhibitors are structurally similar to �-lactam substrates and are not effective inactivators of the class C enzymes, boronic acid transition state inhibitors (BATSIs; chemically dissimilar to �-lactams) were explored to inhibit ADC enzymes. In this study, we used a chiral, carboxy-aryl cephalothin analog BATSI, and the ADC-7 complex was determined at 2.0 � resolution (P21 space group). In the complexed enzyme,the boronic acid makes several canonical interactions. The O1 oxygen is bound in the oxyanion hole, and the R1 amide group makes interactions with conserved residues Asn152 and Gln120. The carboxylate group of the inhibitor mimics the C4' carboxylate found in �-lactams. Asn289, Thr316, and Asn346 commonly comprise the C4' carboxylate recognition residues in class C enzymes. However, in ADC-7, Asn289 is replaced with Glu289 and is pointed out of the active site. Interestingly, in ADC-7 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme, AmpC. The ADC-7/boronic acid complex provides insight into recognition of non-�-lactam inhibitors by ADC enzymes and offers a novel starting point for the structure-based optimization of this class of novel �-lactamase inhibitors against a clinically relevant resistance target.

X-ray crystal structure of the extended-spectrum class C �-lactamase, ADC-7, in apo form and in complex with a boronic acid transition state analog

247th National Meeting (2014)

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