# Biochemical and structural analyses of metallo-β-lactamase VIM-28: impact of substitutions at residues 224 and 228 on substrate profile, stability, and zinc affinity

## TL;DR

The study investigates how specific amino acid changes in the VIM-28 enzyme affect its ability to break down antibiotics, stability, and zinc binding, which could help explain how bacteria develop resistance.

## Contribution

The study reveals how substitutions at positions 224 and 228 in VIM-28 influence substrate specificity, thermal stability, and zinc affinity, offering new insights into MBL evolution.

## Key findings

- Substitutions at positions 224 and 228 in VIM-28 significantly enhance catalytic efficiency for certain antibiotics.
- The R228S substitution improves zinc binding and thermal stability of the enzyme.
- Combined substitutions at positions 224 and 228 increase activity toward ceftazidime.

## Abstract

Metallo-β-lactamases (MBLs) hydrolyze a broad range of β-lactams, including carbapenems. VIM-28, an MBL identified in Pseudomonas aeruginosa, is an H224L/S228R variant of VIM-1 and H224L variant of VIM-4. Compared with VIM-26 (R228S), VIM-28 displayed decreased Km (12.5 for VIM-28 vs 513 μM for VIM-26; 9.66 vs 150 μM) and increased kcat/Km(15.3 vs 1.81 μM−1s−1; 28.6 vs 5.89 μM−1s−1) for ampicillin and cephalothin, respectively. VIM-1, which has a His in position 224 and Ser in position 228, displayed intermediate kinetic values (Km 215 and 77.0 μM; kcat/Km 2.63 and 8.61 μM−1s−1) for ampicillin and cephalothin, respectively, indicating that the presence of a positively charged residue at either position 224 or 228 enhanced substrate interactions. The combined L224H/R228S substitutions in VIM-1 increased the catalytic efficiency of the enzyme for ceftazidime by more than one order of magnitude. These kinetic trends were consistent with the minimum inhibitory concentration (MIC) data, with an eightfold increase in ceftazidime MIC for VIM-1-producing cells. Moreover, relative MIC assay showed that VIM-26 (R228S)-producing cells were more refractory to the addition of chelators than cells producing VIM-28, whereas VIM-4 (L224H)-producing cells showed reduced resistance, suggesting that the residues at positions 224 and 228 influence the metal-binding affinity of the enzyme. Differential scanning fluorimetry assay revealed that the R228S substitution increased the melting temperature of the enzyme, whereas the L224H substitution reduced its thermal stability. VIM-28 exhibited high catalytic efficiency for substrates other than ceftazidime, and the H224L substitution conferred higher zinc-binding affinity and thermal stability compared with VIM-4.

β-Lactam-resistant bacteria, especially carbapenem-resistant strains, pose a major global health threat, often through metallo-β-lactamases (MBLs). To anticipate resistance evolution, we characterized VIM-28, a variant of the widespread VIM-1/VIM-4-type enzymes, focusing on the roles of two variable L10 loop residues. Substitutions at positions 224 and 228 strongly affected substrate specificity, enzyme stability, and zinc affinity. Arg228 was important for carbapenem recognition, while combined substitutions at positions 224 and 228 could enhance activity toward ceftazidime. Notably, the R228S substitution improved zinc binding and thermal stability, supporting enzyme function under zinc-limited host conditions. These findings reveal mechanisms driving MBL diversity and highlight evolutionary strategies sustaining antibiotic resistance.