Site-directed mutagenesis of the metallo-β-lactamase VIM-7 from the opportunistic human pathogenic bacteria Pseudomonas aeruginosa

Abstract

The metallo-β-lactamases (MBLs) are enzymes with the ability to hydrolyse the β-lactam antibiotics. The worldwide emergence of the antibiotic resistant MBLs poses an increasing clinical threat. The VIM enzymes are a growing family of carbapenemases with a wide geographic distribution in Europe, South America and the USA. The VIM-7, the first VIM enzyme to be discovered in the USA, is the most divergent member of the VIM-enzymes with the closest similarity to VIM-1 with a 77% amino acid identity. The VIM-7 has a conserved D120 in the active site, which, in catalysis, plays an important role. The Y224 residue present in the VIM-2, which currently is the most widespread MBL, is though to have an impact on the activity. Three site-directed mutations of the VIM-7, with a previously solved structure, were made; D120A, D120N and H224Y respectively. All three mutants and the VIM-7 wt were sequenced, and the mutants VIM-7 D120A and VIM-7 H224Y with the VIM-7 wt, containing an N-terminal hexahis-tag and cleavage site, were produced, affinity purified and tested in a nitrocefin activity assay. The nitrocefin activity assay showed that the VIM-7 D120A mutant was inactive. In addition, the VIM-7 H224Y mutant was purified using gel filtration. A crystal was obtained of the VIM-7 H224Y mutant, and the crystal structure was solved. Enzyme kinetic studies were performed on the VIM-7 wt and the VM-7 H224Y, which resulted in the VIM-7 H224Y to have a significantly higher catalytic efficiency towards specific antibiotic substrates than the VIM-7 wt. The VIM-7 D120A mutant would not hydrolyse the antibiotic substrate ertapenem, and was not tested with other antibiotics. The inactive VIM-7 D120A and the active VIM-7 H224Y showed that by mutating one single amino acid, the D120 and the H224 residues, it has a significant effect on the activity of the VIM-7.