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Exploring the role of a conserved class A residue in the ?-Loop of KPC-2 ?-lactamase: a mechanism for ceftazidime hydrolysis.

ABSTRACT: Gram-negative bacteria harboring KPC-2, a class A ?-lactamase, are resistant to all ?-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A ?-lactamases and is located in the solvent-exposed ?-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32?128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative ?-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015?0.019 ?m(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of ?-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the ?-loop of KPC-2.

SUBMITTER: Levitt PS

PROVIDER: S-EPMC3442512 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Exploring the role of a conserved class A residue in the Ω-Loop of KPC-2 β-lactamase: a mechanism for ceftazidime hydrolysis.

Levitt Peter S PS Papp-Wallace Krisztina M KM Taracila Magdalena A MA Hujer Andrea M AM Winkler Marisa L ML Smith Kerri M KM Xu Yan Y Harris Michael E ME Bonomo Robert A RA

The Journal of biological chemistry 20120726 38

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (m ...[more]

PMID: 22843686

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Project description:As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.

Project description:The unwelcome evolution of resistance to the advanced generation cephalosporin antibiotic, ceftazidime is hindering the effective therapy of Burkholderia cepacia complex (BCC) infections. Regrettably, BCC organisms are highly resistant to most antibiotics, including polymyxins; ceftazidime and trimethoprim-sulfamethoxazole are the most effective treatment options. Unfortunately, resistance to ceftazidime is increasing and posing a health threat to populations susceptible to BCC infection. We found that up to 36% of 146 tested BCC clinical isolates were nonsusceptible to ceftazidime (MICs ? 8 ?g/ml). To date, the biochemical basis for ceftazidime resistance in BCC is largely undefined. In this study, we investigated the role of the ?-loop in mediating ceftazidime resistance in the PenA ?-lactamase from Burkholderia multivorans, a species within the BCC. Single amino acid substitutions were engineered at selected positions (R164, T167, L169, and D179) in the PenA ?-lactamase. Cell-based susceptibility testing revealed that 21 of 75 PenA variants engineered in this study were resistant to ceftazidime, with MICs of >8 ?g/ml. Under steady-state conditions, each of the selected variants (R164S, T167G, L169A, and D179N) demonstrated a substrate preference for ceftazidime compared to wild-type PenA (32- to 320-fold difference). Notably, the L169A variant hydrolyzed ceftazidime significantly faster than PenA and possessed an ?65-fold-lower apparent Ki (Kiapp) than that of PenA. To understand why these amino acid substitutions result in enhanced ceftazidime binding and/or turnover, we employed molecular dynamics simulation (MDS). The MDS suggested that the L169A variant starts with the most energetically favorable conformation (-28.1 kcal/mol), whereas PenA possessed the most unfavorable initial conformation (136.07 kcal/mol). In addition, we observed that the spatial arrangement of E166, N170, and the hydrolytic water molecules may be critical for enhanced ceftazidime hydrolysis by the L169A variant. Importantly, we found that two clinical isolates of B. multivorans possessed L169 amino acid substitutions (L169F and L169P) in PenA and were highly resistant to ceftazidime (MICs ? 512 ?g/ml). In conclusion, substitutions in the ?-loop alter the positioning of the hydrolytic machinery as well as allow for a larger opening of the active site to accommodate the bulky R1 and R2 side chains of ceftazidime, resulting in resistance. This analysis provides insights into the emerging phenotype of ceftazidime-resistant BCC and explains the evolution of amino acid substitutions in the ?-loop of PenA of this significant clinical pathogen.

Project description:The spread of ?-lactamases that hydrolyze penicillins, cephalosporins and carbapenems among Gram-negative bacteria has limited options for treating bacterial infections. Initially, Klebsiella pneumoniae carbapenemase-2 (KPC-2) emerged as a widespread carbapenem hydrolyzing ?-lactamase that also hydrolyzes penicillins and cephalosporins but not cephamycins and ceftazidime. In recent years, single and double amino acid substitution variants of KPC-2 have emerged among clinical isolates that show increased resistance to ceftazidime. Because it confers multi-drug resistance, KPC ?-lactamase is a threat to public health. In this study, the evolution of KPC-2 function was determined in nine clinically isolated variants by examining the effects of the substitutions on enzyme kinetic parameters, protein stability and antibiotic resistance profile. The results indicate that the amino acid substitutions associated with KPC-2 natural variants lead to increased catalytic efficiency for ceftazidime hydrolysis and a consequent increase in ceftazidime resistance. Single substitutions lead to modest increases in catalytic activity while the double mutants exhibit significantly increased ceftazidime hydrolysis and resistance levels. The P104R, V240G and H274Y substitutions in single and double mutant combinations lead to the largest increases in ceftazidime hydrolysis and resistance. Molecular modeling suggests that the P104R and H274Y mutations could facilitate ceftazidime hydrolysis through increased hydrogen bonding interactions with the substrate while the V240G substitution may enhance backbone flexibility so that larger substrates might be accommodated in the active site. Additionally, we observed a strong correlation between gain of catalytic function for ceftazidime hydrolysis and loss of enzyme stability, which is in agreement with the 'stability-function tradeoff' phenomenon. The high Tm of KPC-2 (66.5°C) provides an evolutionary advantage as compared to other class A enzymes such as TEM (51.5°C) and CTX-M (51°C) in that it can acquire multiple destabilizing substitutions without losing the ability to fold into a functional enzyme.

Project description:Beta-lactamase-mediated antibiotic resistance continues to challenge the contemporary treatment of serious bacterial infections. The KPC-2 beta-lactamase, a rapidly emerging gram-negative resistance determinant, hydrolyzes all commercially available beta-lactams, including carbapenems and beta-lactamase inhibitors; the amino acid sequence requirements responsible for this versatility are not yet known. To explore the bases of beta-lactamase activity, we conducted site saturation mutagenesis at Ambler position 237. Only the T237S variant of the KPC-2 beta-lactamase expressed in Escherichia coli DH10B maintained MICs equivalent to those of the wild type (WT) against all of the beta-lactams tested, including carbapenems. In contrast, the T237A variant produced in E. coli DH10B exhibited elevated MICs for only ampicillin, piperacillin, and the beta-lactam-beta-lactamase inhibitor combinations. Residue 237 also plays a novel role in inhibitor discrimination, as 11 of 19 variants exhibit a clavulanate-resistant, sulfone-susceptible phenotype. We further showed that the T237S variant displayed substrate kinetics similar to those of the WT KPC-2 enzyme. Consistent with susceptibility testing, the T237A variant demonstrated a lower k(cat)/K(m) for imipenem, cephalothin, and cefotaxime; interestingly, the most dramatic reduction was with cefotaxime. The decreases in catalytic efficiency were driven by both elevated K(m) values and decreased k(cat) values compared to those of the WT enzyme. Moreover, the T237A variant manifested increased K(i)s for clavulanic acid, sulbactam, and tazobactam, while the T237S variant displayed K(i)s similar to those of the WT. To explain these findings, a molecular model of T237A was constructed and this model suggested that (i) the hydroxyl side chain of T237 plays an important role in defining the substrate profile of the KPC-2 beta-lactamase and (ii) hydrogen bonding between the hydroxyl side chain of T237 and the sp(2)-hybridized carboxylate of imipenem may not readily occur in the T237A variant. This stringent requirement for selected cephalosporinase and carbapenemase activity and the important role of T237 in inhibitor discrimination in KPC-2 are central considerations in the future design of beta-lactam antibiotics and inhibitors.

Dataset Information

Exploring the role of a conserved class A residue in the ?-Loop of KPC-2 ?-lactamase: a mechanism for ceftazidime hydrolysis.

Publications

Exploring the role of a conserved class A residue in the Ω-Loop of KPC-2 β-lactamase: a mechanism for ceftazidime hydrolysis.

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