Mutational Analysis of VIM-2 Reveals an Essential Determinant for Metallo-β-Lactamase Stability and Folding

Abstract

Metallo-␤-lactamase (MBL)-producing bacteria are emerging worldwide and represent a formidable threat to the efficacy of relevant ␤-lactams, including carbapenems, expanded-spectrum cephalosporins, and ␤-lactamase inactivator/␤-lactam combinations. VIM-2 is currently the most widespread MBL and represents a primary target for MBL inhibitor research, the clinical need for which is expected to further increase in the future. Using a saturation mutagenesis approach, we probed the importance of four residues (Phe-61, Ala-64, Tyr-67, and Trp-87) located close to the VIM-2 active site and putatively relevant to the enzyme activity based on structural knowledge of the enzyme and on structure-activity relationships of the subclass B1 MBLs. The ampicillin MIC values shown by the various mutants were affected very differently depending on the randomized amino acid position. Position 64 appeared to be rather tolerant to substitution, and kinetic studies showed that the A64W mutation did not significantly affect substrate hydrolysis or binding, representing an important difference from IMP-type enzymes. Phe-61 and Tyr-67 could be replaced with several amino acids without the ampicillin MIC being significantly affected, but in contrast, Trp-87 was found to be critical for ampicillin resistance. Further kinetic and biochemical analyses of W87A and W87F variants showed that this residue is apparently important for the structure and proper folding of the enzyme but, surprisingly, not for its catalytic activity. These data support the critical role of residue 87 in the stability and folding of VIM-2 and might have strong implications for MBL inhibitor design, as this residue would represent an ideal target for interaction with small molecules.

Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15

“…Our results were consistent with previous findings showing that A 77 V played an important role in the phenotype of expanded-spectrum cephalosporin resistance, in which A 77 V increased resistance to cefotaxime and ceftazidime when associated with P 167 and G 240 in the background of CTX-M-10 (16). Collectively, these data suggested that, albeit being located distal to the active site, A 77 V plays an important role on the catalytic activity of CTX-Ms through stabilizing the structure of the enzymes, which has also been reported in other ␤-lactamases (17).…”

Extensively Drug-Resistant Pseudomonas aeruginosa Isolates Containing bla VIM-2 and Elements of Salmonella Genomic Island 2: a New Genetic Resistance Determinant in Northeast Ohio

“…In order to assess VIM-2 expression, rabbit polyclonal antibodies were raised against purified VIM-2 ␤-lactamase by New England Peptide and isolated from serum using protein G column purification (Sigma Genosys, The Woodlands, TX). Sample preparation, immunoblotting, and development were performed as previously reported (29,30).…”

Evolution to carbapenem-hydrolyzing activity in noncarbapenemase class D β-lactamase OXA-10 by rational protein design

“…The reaction conditions used were the following: an initial denaturation step at 96°C for 2 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 48°C for 1 min, extension at 72°C for 1 min and 30 s, and a final extension step at 72°C for 20 min. The 760-bp amplification product was subcloned in the EcoRI and BamHI restriction sites of pLB-II vector (18), to obtain the pLBII-OXA-10 plasmid. The OXA-10 laboratory variants were constructed by overlapping PCR with partially overlapping primers (Table 5), as previously described (19), using the pLBII-OXA10 plasmid as the template.…”