Vibrio vulnificus is a pathogen that accounts for one of the highest mortality rates and is responsible for most reported seafood-related illnesses and deaths worldwide. Owing to the threats of pathogens with β-lactamase activity, it is important to identify and characterize β-lactamases with clinical significance. In this study, the protein sequence of the metallo-β-lactamase (MBL) fold metallohydrolase from V. vulnificus (designated Vmh) was analyzed, and its oligomeric state, β-lactamase activity, and metal binding ability were determined. BLASTp analysis indicated that the V. vulnificus Vmh protein showed no significant sequence identity with any experimentally identified Ambler class B MBLs or enzymes containing the MBL protein fold; it was also predicted to have a signal peptide of 19 amino acids at its N terminus and an MBL protein fold from amino acid residues 23 to 216. Recombinant V. vulnificus Vmh protein was overexpressed and purified. Analytical ultracentrifugation and electrospray ionization-mass spectrometry (MS) data demonstrated its monomeric state in an aqueous solution. Recombinant V. vulnificus Vmh protein showed broad degrading activities against β-lactam antibiotics, such as penicillins, cephalosporins, and imipenems, with kcat/Km values ranging from 6.23 × 102 to 1.02 × 104 M-1 s-1. The kinetic reactions of this enzyme exhibited sigmoidal behavior, suggesting the possibility of cooperativity. Zinc ions were required for the enzyme activity, which was abolished by adding the metal chelator EDTA. Inductively coupled plasma-MS indicated that this enzyme might bind two zinc ions per molecule as a cofactor.
Keywords: ICP-MS; LC-MS; Vibrio vulnificus; analytical ultracentrifugation; cooperativity; metallo-β-lactamase (MBL) protein fold; β-lactam antibiotics; β-lactamases.