{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:42:23Z","timestamp":1785415343630,"version":"3.56.0"},"reference-count":38,"publisher":"American Society for Microbiology","issue":"3","license":[{"start":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T00:00:00Z","timestamp":1772496000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T00:00:00Z","timestamp":1772496000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,3,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Metallo-\u03b2-lactamases (MBLs) hydrolyze a broad range of \u03b2-lactams, including carbapenems. VIM-28, an MBL identified in\n                      <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                      , is an H224L\/S228R variant of VIM-1 and H224L variant of VIM-4. Compared with VIM-26 (R228S), VIM-28 displayed decreased\n                      <jats:italic toggle=\"yes\">K<\/jats:italic>\n                      <jats:sub>m<\/jats:sub>\n                      (12.5 for VIM-28 vs 513 \u03bcM for VIM-26; 9.66 vs 150 \u03bcM) and increased\n                      <jats:italic toggle=\"yes\">k<\/jats:italic>\n                      <jats:sub>cat<\/jats:sub>\n                      \/\n                      <jats:italic toggle=\"yes\">K<\/jats:italic>\n                      <jats:sub>m<\/jats:sub>\n                      (15.3 vs 1.81 \u03bcM\n                      <jats:sup>\u22121<\/jats:sup>\n                      s\n                      <jats:sup>\u22121<\/jats:sup>\n                      ; 28.6 vs 5.89 \u03bcM\n                      <jats:sup>\u22121<\/jats:sup>\n                      s\n                      <jats:sup>\u22121<\/jats:sup>\n                      ) for ampicillin and cephalothin, respectively. VIM-1, which has a His in position 224 and Ser in position 228, displayed intermediate kinetic values (\n                      <jats:italic toggle=\"yes\">K<\/jats:italic>\n                      <jats:sub>m<\/jats:sub>\n                      215 and 77.0 \u03bcM;\n                      <jats:italic toggle=\"yes\">k<\/jats:italic>\n                      <jats:sub>cat<\/jats:sub>\n                      \/\n                      <jats:italic toggle=\"yes\">K<\/jats:italic>\n                      <jats:sub>m<\/jats:sub>\n                      2.63 and 8.61 \u03bcM\n                      <jats:sup>\u22121<\/jats:sup>\n                      s\n                      <jats:sup>\u22121<\/jats:sup>\n                      ) 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.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\u03b2-Lactam-resistant bacteria, especially carbapenem-resistant strains, pose a major global health threat, often through metallo-\u03b2-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.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02788-25","type":"journal-article","created":{"date-parts":[[2026,2,9]],"date-time":"2026-02-09T13:00:46Z","timestamp":1770642046000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Biochemical and structural analyses of metallo-\u03b2-lactamase VIM-28: impact of substitutions at residues 224 and 228 on substrate profile, stability, and zinc affinity"],"prefix":"10.1128","volume":"14","author":[{"given":"Hiromu","family":"Sato","sequence":"first","affiliation":[{"name":"Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences","place":["Niigata, 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