Amino acid changes at the VIM-48 C-terminus result in increased carbapenem resistance, enzyme activity and protein stability

Objectives Since the identification of VIM-1, point substitutions resulting in variants with differing hydrolytic activities have occurred, driving the evolution of the VIM enzymes. We previously detected a novel variant, VIM-48, containing 11 successive amino acid (aa) alterations in the C-terminal region compared with VIM-2. Single aa substitutions significantly change enzyme properties, but the effects of successive aa alterations have not previously been studied. Herein, we aimed to investigate the sequence and biochemical characteristics of VIM-48, including the role of the 11 successive aa substitutions. Methods VIM-48, VIM-2 and a truncated VIM-D() mutant missing 11 aa at the C-terminus relative to VIM-48 were characterized by antimicrobial susceptibility testing, protein expression and purification, determination of kinetic parameters, and homology modelling. Protein secondary structure and thermal stability measurements were also performed using circular dichroism spectral analysis. Results Compared with bla(VIM-2), bla(VIM-48) conferred higher resistance to carbapenems. VIM expression in Pseudomonas putida resulted in higher MICs than in E. coli. VIM-48 demonstrated increased hydrolytic activity against carbapenems relative to VIM-2, while VIM-D() had significantly decreased catalytic efficiency compared with VIM-2 and VIM-48 as a result of aa deletion. In addition, secondary structure analysis revealed that VIM-48 had the greatest proportion of -helices among the tested enzymes, corresponding to increased thermostability, while VIM-D() had the lowest proportion of -helices and decreased thermostability. Conclusions VIM-48 has increased enzymatic activity and thermostability and increases host -lactam resistance. Observed changes in the secondary structure of VIM-48 resulted from successive aa alterations. Therefore, VIM evolution likely occurs via both single and successive aa substitutions.