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

This work investigated the molecular events driving the evolution of the CTX-M-type beta-lactamases by the use of 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 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 (PL)-L-67 and (LP)-P-261 changes in a CTX-M-14 fragment could completely abolish the activity of the enzyme on all antibiotics tested. Structural analysis showed that L-216 was located in the active-site beta sheet and might interact with the adjacent hydrophobic residues to stabilize the active-site beta sheet and maintain the integrity of the enzyme active site. Likewise, a single amino acid substitution, (EK)-K-64, was found to exhibit a significant suppressive effect on CTX-M-15 activity. Structural analysis showed that E-64 might form a salt bridge with R-44, 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.