期刊
PLOS ONE
卷 8, 期 9, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0073123
关键词
-
资金
- National Science Foundation [0449080]
- New York State Office of Science, Technology and Academic Research (NYSTAR)
- NYSTAR Distinguished Faculty Award
- Directorate For Engineering
- Div Of Chem, Bioeng, Env, & Transp Sys [0449080] Funding Source: National Science Foundation
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 beta-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 beta-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to beta-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., broken vertical bar 105F) that appears to widen access to the active site by 3.6 angstrom while also stabilizing the reorganized topology. In light of these findings, we propose that broken vertical bar 105 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据