His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

Abstract

b Metallo-␤-lactamases (MBLs) are the causative mechanism for resistance to ␤-lactams, including carbapenems, in many Gramnegative 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 roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T m ) of 48.5°C, compared to 55.3°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 enhanced activity compared to wild-type tVIM-7, and a slightly higher T m (57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T m , 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all 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 toward the positively charged cephalosporins like cefepime and ceftazidime.

Structural and biochemical characterization of VIM‐26 shows that Leu224 has implications for the substrate specificity of VIM metallo‐β‐lactamases

“…In addition there are two stabilizing hydrogen bonds in VIM‐2 involving Tyr224 . We later confirmed this through structural and kinetic studies of a His224Tyr mutation of VIM‐7 where we could show that the mutant was more active against positively charged cephalosporins compared to the wild‐type VIM‐7 . In a VIM‐2 thiol based inhibitor complex it was found that both Arg228 and Asn233 are important for inhibitor binding and recognition .…”

“…Compared to the other VIM variants, the overall trend was that VIM‐26 and periplasmic purified VIM‐7 were more efficient penicillinases than VIM‐1 and VIM‐2 (Table ). In contrast both VIM‐1 (all cephalosporins) and to a certain degree VIM‐2 (ceftazidime and cefoxitin) were more efficient cephalosporinases than VIM‐26 and VIM‐7.…”

“…VIM‐26 differs from VIM‐1 by a single residue, namely histidine 224, which has been substituted with a leucine . This residue is part of the L3 loop involved in the R2 substrate binding site, and plays an important role in catalysis . All the VIM‐26 structures showed an open binding site for the R2 part of the substrates (Fig.…”

Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding

“…The tyrosine at 224 position in VIM-2 contributes to enhanced binding of positively charged substrates, such as ceftazidime and cefepime, compared to the arginine and lysine present at this position in TMB-1, NDM-1, and GIM-1, as shown for the VIM-7 H224Y mutant (36).…”

“…Residue 228 has not been shown to be involved in the binding of penicillin substrates with small R2 groups; however, the residue seems to affect neighboring residues in the active site, including the L3 loop, which again affects the binding of penicillin substrates. Residue 228 is located adjacent to the active-site Zn2 in the so-called R2 binding site (36) and is a part of the MBL L3 loop. Substitutions at position 228 have been shown to be tolerated for different substrates (37) but have also been found to affect the substrate specificity of, e.g., GIM-1 (25).…”