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The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for β-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P → S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower Km values, suggesting that the P → S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P → S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases but may also provide a map for β-lactam improvement.

β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target.

The most common mechanism responsible for β-lactam resistance in Gram-negative bacteria is the production of β-lactamases (EC 3.5.2.6) (1). At present, there are more than 850 different β-lactamases described in nature (http://www.lahey.org/Studies/). As a family of proteins, β-lactamases are grouped into four main classes (i.e., A to D) based upon amino acid sequence homology (2). Classes A, C, and D employ serine in the active site as the reactive nucleophile, while class B β-lactamases use a single or pair of metal ions (Zn2+) to catalyze the opening of the β-lactam ring. Both serine and metallo-β-lactamases make use of a strategically positioned water molecule to hydrolyze the lactam bond (3-5). In general, class A, C, and D β-lactamases follow a three-step reaction mechanism that is represented as follows: E+S⇌k−1k1E:S→k2E-S→H2Ok3E+P (Eq. 1) Here, E is the β-lactamase enzyme, S is the β-lactam substrate, E:S is the Henri-Michaelis complex, E-S is the acyl enzyme, and P is the inactivated β-lactam. The rate constants for each step are designated by k1, k-1, k2, and k3. Usually, class A and C β-lactamases are found in the Enterobacteriaceae. Acquired class A enzymes, often carried on mobile plasmids, are common in Escherichia coli and Klebsiella pneumoniae, are constitutively expressed and confer resistance to penicillins and narrow-spectrum cephalosporins (1). Class C, or AmpC-type β-lactamases (AmpCs), are encoded by chromosomal genes in many Gram-negative pathogens (e.g., Citrobacter freundii, Enterobacter spp., and Pseudomonas aeruginosa), are inducible, and confer resistance to narrow and extended-spectrum cephalosporins. Most recently, an increasing number of AmpC β-lactamase genes are being discovered on plasmids that also spread by horizontal transfer (6-8). Unlike class A enzymes, AmpCs are poorly inhibited by the commercially available β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) (7). Among class C enzymes, CMY-type β-lactamases represent the most frequently detected plasmid encoded AmpCs (pAmpCs) (8-13). Unfortunately, we know little regarding the ability of clinically important CMY enzymes to hydrolyze different classes of β-lactam antibiotics (14-16). Furthermore, the activity of novel β-lactamase inhibitors against the CMY type pAmpCs has not yet been fully studied (17). In class A β-lactamases, single amino acid substitutions (primarily, but not exclusively, at Ambler positions Gly238 on the b3 β-strand and Arg164 or Asp179 in the Ω loop) remodel a “broad-spectrum” β-lactamase into an extended-spectrum β-lactamase (ESBL) (18-20). The bacterium possessing a class A ESBL becomes resistant to ceftazidime and/or cefotaxime. However, these changes come at a “price” (18, 21). Among class A ESBLs, one uniformly detects a decrease in penicillin resistance (lower MICs) and an increase in the susceptibility to β-lactamase inhibitors and carbapenems (18, 21). Herein, we analyzed the biochemical properties of a novel CMY-2-like pAmpC (i.e., CMY-32) cloned from an E. coli isolate found in the clinic. In comparison with CMY-2 (wild type, WT), we determined that CMY-32 possesses a single amino acid substitution in the Ω loop (i.e., Gly214Glu) resulting in significant changes in the resistance phenotype and hydrolytic profile (making this β-lactamase an “extended-spectrum type”). We also evaluated the mechanisms by which carbapenems, a monobactam antibiotic aztreonam, and the novel monobactam derivative, {"type":"entrez-protein","attrs":{"text":"BAL29880","term_id":"359272361"}}BAL29880, react and inhibit CMY-2 and CMY-32 enzymes. Our data reveal the impact of single amino acid substitutions on protein evolution in this emerging family of resistance enzymes and highlight the manner in which carbapenems and monobactams inactivate class C enzymes.