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The Class D beta-lactamase family: residues governing the maintenance and diversity of function.

ABSTRACT: Class D ?-lactamases, a major source of bacterial resistance to ?-lactam antibiotic therapies, represent a distinct subset of the ?-lactamase superfamily. They share a serine hydrolase mechanism with Classes A/C vs. Class B. Further understanding of their sequence-structure-function relationships would benefit efforts to design a new generation of antibiotics as well as to predict evolutionary mechanisms in response to such therapies. Here we describe analyses based on our high-resolution multiple sequence alignment and phylogenetic tree of ?80 Class D ?-lactamases that leverage several 3D structures of these enzymes. We observe several sequence clusters on the phylogenetic tree, some that are species specific while others include several species from ?-, ?- and ?-proteobacteria. Residues characteristic of a specific cluster were identified and shown to be located just outside the active site, possibly modulating the function of the catalytic residues to facilitate reactions with specific types of ?-lactams. Most significant was the discovery of a likely disulfide bond in a large group composed of ?-, ?- and ?-proteobacteria that would contribute to enzyme stability and hence bacterial viability under antibiotic assault. A network of co-evolving residues was identified which suggested the importance of maintaining a surface for binding a highly conserved Phe69.

SUBMITTER: Szarecka A

PROVIDER: S-EPMC3170078 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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The Class D beta-lactamase family: residues governing the maintenance and diversity of function.

Szarecka Agnieszka A Lesnock Kimberly R KR Ramirez-Mondragon Carlos A CA Nicholas Hugh B HB Wymore Troy T

Protein engineering, design & selection : PEDS 20110822 10

Class D β-lactamases, a major source of bacterial resistance to β-lactam antibiotic therapies, represent a distinct subset of the β-lactamase superfamily. They share a serine hydrolase mechanism with Classes A/C vs. Class B. Further understanding of their sequence-structure-function relationships would benefit efforts to design a new generation of antibiotics as well as to predict evolutionary mechanisms in response to such therapies. Here we describe analyses based on our high-resolution multip ...[more]

PMID: 21859796

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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.

Dataset Information

The Class D beta-lactamase family: residues governing the maintenance and diversity of function.

Publications

The Class D beta-lactamase family: residues governing the maintenance and diversity of function.

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