Comparative insight into the roles of the non active-site residues E169 and N173 in imparting the beta-lactamase activity of CTX-M-15

Abstract

Abstract CTX-M-15 is a major extended-spectrum beta-lactamase disseminated throughout the globe. The roles of amino acids present in the active-site are widely studied though little is known about the role of the amino acids lying at the close proximity of the CTX-M-15 active-site. Here, by using site-directed mutagenesis we attempted to decipher the role of individual amino acids lying outside the active-site in imparting the beta-lactamase activity of CTX-M-15. Based on the earlier evidence, three amino acid residues namely, Glu169, Asp173 and Arg277 were substituted with alanine. The antibiotic susceptibility of E. coli cells harboring E169A and N173A substituted CTX-M-15 were enhanced by ∼ >32 fold for penicillins and ∼ 4–32 fold for cephalosporins, in comparison to CTX-M-15. However, cells carrying CTX-M-15_R277A did not show a significant difference in antibiotic susceptibility as compared to the wild-type. Further, the catalytic efficiency of the purified CTX-M-15_E169A and CTX-M-15_N173A were compromised when compared with the efficient beta-lactam hydrolysis of purified CTX-M-15. Moreover, the thermal stability of the mutated proteins CTX-M-15_E169A and CTX-M-15_N173A were reduced as compared to the wild type CTX-M-15. Therefore, we conclude that E169 and N173 are crucial non-active-site amino acids that are able to govern the CTX-M-15 activity.