4d1t
From Proteopedia
High resolution structure of native tVIM-7 from Pseudomonas aeruginosa
Structural highlights
FunctionPublication Abstract from PubMedMetallo-beta-lactamases (MBLs) are the causative mechanism for resistance to beta-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study the importance of residues Asp120, Phe218 and His224 in the most divergent VIM-variant, VIM-7, was investigated to better understand the role of these residues among VIM-enzyme through mutations, enzyme kinetics, crystal structures, thermostability and docking experiments. The tVIM-7-D120A mutant was enzymatically inactive and structure showed the presence of only the Zn1 ion. The mutant was less thermostable with a melting temperature (Tm) of 48.5 degrees C compared to 55.3 degrees C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84 and Arg121. The tVIM-7-F218Y mutant had an enhanced activity compared wild type tVIM-7, and a slightly higher Tm (57.1 degrees C) was observed most likely due to the hydrogen bonding cluster Further, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (Tm 62.9 degrees C) and an increased enzymatic activity compared to the wild type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y and VIM-7-F218Y found the side-chain conformation of residue 224 and Arg228 in the L3 loop, and Tyr67 in the L1 loop, all to influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly towards the positively charged cephalosporins like cefepime and ceftazidime. His224 alters the R2 drug binding site and Phe218 influences the catalytic efficiency in the metallo-beta-lactamase VIM-7.,Leiros HK, Skagseth S, Edvardsen KS, Lorentzen MS, Bjerga GE, Leiros I, Samuelsen O Antimicrob Agents Chemother. 2014 Jun 9. pii: AAC.02735-13. PMID:24913158[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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