Functional characterization of CTX-M-14 and CTX-M-15 {beta}-lactamase by in vitro DNA shuffling [PublishAheadOfPrint]

This work investigated the molecular events driving the evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the bla_CTX-M-14 and bla_CTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet active hybrids possessed fewer amino acid substitutions than those that were inactive, suggesting that point mutations in the constructs, rather than re-shuffling 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 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.

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