On functional and structural heterogeneity of VIM-type metallo-beta-lactamases

Abstract

The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded approximately 80 mg of protein per litre of culture. The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency. Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in K(m) values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour.

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

“…An OXA-10-producing E. coli laboratory strain was obtained following PCR amplification of the bla OXA-10 ORF with specific primers (Table 5) with the Expand High Fidelity PCR system (Roche Biochemicals), as previously described (17). 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.…”

Role of Zinc Content on the Catalytic Efficiency of B1 Metallo β-Lactamases

“…9-14 Bacteroides fragilis (CcrA), 15-17 Elizabethkingia meningoseptica (BlaB), 18,19 as well as the transferable VIM, IMP, SPM and GIM-type enzymes. [20][21][22][23][24][25][26] includes the CphA and imiS lactamases from Aeromonas species. 27-29 Subclass B3, along with the extensively characterized enzyme L1 from Stenotrophomonas maltophilia ,30-33 includes enzymes from environmental bacteria, such as CAU-1 from Caulobacter crescentus 34 and THIN-B from Janthinobacterium lividum , 35 and from opportunistic pathogens like FEZ-1 from Legionella gormanii ,36-38 and GOB from E. meningoseptica.…”

Targeting clinically-relevant metallo-β-lactamases: from high-throughput docking to broad-spectrum inhibitors

“…The MBL final concentration in the assays ranged 2.5-40 nM. The K i value was determined as previously described, using a competitive model of inhibition 38 .…”