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Directed evolution of Mycobacterium tuberculosis ?-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

ABSTRACT: Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the ?-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A ?-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to ?-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a 'gatekeeper' residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.

SUBMITTER: Feiler C

PROVIDER: S-EPMC3762836 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

Feiler Christian C Fisher Adam C AC Boock Jason T JT Marrichi Matthew J MJ Wright Lori L Schmidpeter Philipp A M PA Blankenfeldt Wulf W Pavelka Martin M DeLisa Matthew P MP

PloS one 20130904 9

Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme lib ...[more]

PMID: 24023821

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Project description:BackgroundTrehalases have potential applications in several fields, including food additives, insecticide development, and transgenic plant. In the present study, we focused on a trehalase from the marine bacterium Zunongwangia sp., which hydrolyzes trehalose to glucose.ResultsA novel gene, treZ (1590 bp) encoding an α, α-trehalase of 529 amino acids was cloned from Zunongwangia sp., and TreZ was found to have an optimal activity at 50 °C and pH 6. The activity of TreZ was increased by the presence of NaCl, showing the highest activity (136 %) at 1 M NaCl. A variant C4 with improved catalytic activity was obtained by error-prone PCR and followed by a 96-well plate high-throughput screening. The variant C4 with two altered sites (Y227H, and R442G) displayed a 3.3 fold increase in catalytic efficiency (k cat/K m, 1143.40 mmol(-1) s(-1)) compared with the wild type enzyme (265.91 mmol(-1) s(-1)). In order to explore the contribution of the mutations found in variant C4 to the increased catalytic activity, two mutants Y227H and R442G were constructed by site-directed mutagenesis. The results showed that the catalytic efficiencies of Y227H and R442G were 416.78 mmol(-1) s(-1) and 740.97 mmol(-1) s(-1), respectively, indicating that both mutations contributed to the increased catalytic efficiency of variant C4. The structure modeling and substrate docking revealed that the substitution Y227H enlarged the shape of the binding pocket, to improve the binding of the substrate and the release of the products; while the substitution R442G reduced the size of the side chain and decreased the steric hindrance, which contributed to channel the substrate into the active cavity easier and promote the release of the product.ConclusionIn this study, a novel trehalase was cloned, purified, characterized, and engineered. A variant C4 with dramatically improved catalytic activity was obtained by directed evolution, and the mutation sites Y227H and R442G were found to play a significant role in the catalytic efficiency. The overall results provide useful information about the structure and function of trehalase.

Dataset Information

Directed evolution of Mycobacterium tuberculosis ?-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

Publications

Directed evolution of Mycobacterium tuberculosis β-lactamase reveals gatekeeper residue that regulates antibiotic resistance and catalytic efficiency.

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