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Evolution of Broad Spectrum beta-Lactam Resistance in an Engineered Metallo-beta-lactamase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 4, 2314-2324 p.Article in journal (Refereed) Published
Abstract [en]

The extensive use and misuse of antibiotics during the last seven decades has led to the evolution and global spread of a variety of resistance mechanisms in bacteria. Of high medical importance are beta-lactamases, a group of enzymes inactivating beta-lactam antibiotics. Metallo-beta-lactamases (MBLs) are particularly problematic because of their ability to act on virtually all classes of beta-lactam antibiotics. An engineered MBL (evMBL9) characterized by low level activity with several beta-lactam antibiotics was constructed and employed as a parental MBL in an experiment to examine how an enzyme can evolve toward increased activity with a variety of beta-lactam antibiotics. We designed and synthesized a mutant library in which the substrate activity profile was varied by randomizing six active site amino acid residues. The library was expressed in Salmonella typhimurium, clones with increased resistance against seven different beta-lactam antibiotics (penicillin G, ampicillin, cephalothin, cefaclor, cefuroxime, cefoperazone, and cefotaxime) were isolated, and the MBL variants were characterized. For the majority of the mutants, bacterial resistance was significantly increased despite marked reductions in both mRNA and protein levels relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of the mutant enzymes were generalists, conferring increased resistance against most of the examined beta-lactams.

Place, publisher, year, edition, pages
2013. Vol. 288, no 4, 2314-2324 p.
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Medical and Health Sciences Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-196042DOI: 10.1074/jbc.M112.430199ISI: 000314211500021OAI: oai:DiVA.org:uu-196042DiVA: diva2:609035
Available from: 2013-03-04 Created: 2013-03-04 Last updated: 2017-12-06Bibliographically approved

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Sun, SongMannervik, BengtAndersson, Dan I.

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