Evolution of increased ß-lactam resistance in an engineered Metallo-ß-lactamase
(English)Manuscript (preprint) (Other academic)
The extensive use and misuse of antibiotics during the last 60 years has led to the evolution and global spread of a variety of resistance mechanisms. Of high medical importance are ß-lactamases, a group of enzymes that can hydrolyze the ß-lactam ring present in all ß-lactam antibiotics. Metallo-ß-lactamases (MBLs) are particularly problematic due to their ability to hydrolyze virtually all classes of ß-lactam antibiotics. A novel MBL (evMBL9) with low-level resistance against ß-lactam antibiotics was designed and employed as the ancestral MBL during an experiment to examine how an enzyme evolved towards increased resistance. We designed and synthesized a mutant library in which the substrate binding profile was varied by randomizing six amino acid residues. Mutants with increased resistance against seven different ß-lactam antibiotics (penicillin G, ampicillin, cefalotin, cefaclor, cefuroxime, cefoperazone and cefotaxime) were isolated and characterized. For the majority of mutants, in spite of their significantly increased resistance, both mRNA and protein levels were reduced (up to >20 fold) 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 mutant enzymes became generalists, conferring increased resistance against most of the examined ß-lactams. The increased resistance and decreased protein level suggest that the improved hydrolysis in these novel MBLs is associated with decreased protein stability.
Metallo-beta-lactamase, directed protein evolution
Other Biological Topics
IdentifiersURN: urn:nbn:se:uu:diva-172785OAI: oai:DiVA.org:uu-172785DiVA: diva2:515765