Functional Characterization of CTX-M-14 and CTX-M-15 β-Lactamases by In Vitro DNA Shuffling

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journal

2 Scopus Citations
View graph of relations

Author(s)

Detail(s)

Original languageEnglish
Article numbere00891
Journal / PublicationAntimicrobial Agents and Chemotherapy
Volume61
Issue number12
Online published22 Nov 2017
Publication statusPublished - Dec 2017
Externally publishedYes

Abstract

This work investigated the molecular events driving the evolution of the CTX-M-type β-lactamases by the use of DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet hybrids that were active possessed fewer amino acid substitutions than those that were inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. For example, the P67L and L261P changes in a CTX-M-14 fragment could completely abolish the activity of the enzyme on all antibiotics tested. Structural analysis showed that L216 was located in the active-site β sheet and might interact with the adjacent hydrophobic residues to stabilize the active-site β sheet and maintain the integrity of the enzyme active site. Likewise, a single amino acid substitution, E64K, was found to exhibit a significant suppressive effect on CTX-M-15 activity. Structural analysis showed that E64 might form a salt bridge with R44, disruption of which might affect CTX-M-15 activity. Further analysis of the structure-function relationship of a range of mutant enzymes confirmed that, as can be expected, unstable enzymes lose their activity and avoid selective events. These findings suggest that the distal pockets could also contribute to the activity of the enzymes and may be regarded as alternative targets for inhibitor development.

Research Area(s)

  • CTX-M-14, CTX-M-15, DNA shuffling, Evolution, Hybrid enzyme