Metallo-beta-lactamase CphA evolving into more efficient hydrolases through gene mutation is a novel pathway for the resistance of super bacteria

The evolution of metallo-beta-lactamase CphA in discontinuous gradient concentration of imipenem was investigated in this work. The results suggested that single-base mutations K218R, K249T, K249M, Q253H, and a frameshift mutation M1 were observed. Compared with wild type, the minimum inhibitory concentration (MICs) of K249T, K249M, and M1 increased by at least 128 times and that of K218R increased by 64 times. And the catalytic efficiency increased by 312% and 653%, respectively. It is speculated from the details of the structural changes revealed by molecular dynamics simulations that the carbon skeleton migration caused by the outward motion of the loop 3 in the mutant may have significantly increased the cavity volume of the binding pocket, which is more conducive to the entry and expulsion of imipenem and its hydrolytic product. And the conformational change of the TDRAGGN (71-77) is located at the bottom of the binding pocket from order alpha-helix to disorder random coil enabled the binding pocket to be more conducive to accommodate and hold the imipenem respectively. All these indicated that during the repeated drug resistance, the wild-type achieved gene mutations and conformational change and evolved to the mutant enzymes with a more delicate structure and stronger hydrolysis ability.