Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo- -lactamases

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.

Section: R-sym ϭ {[⌺ H ⌺ I | I I (H) ϫ ͗I(h)͘|]/[⌺ H ⌺ I I(h)]} Whersupporting

confidence: 86%

Section: R-sym ϭ {[⌺ H ⌺ I | I I (H) ϫ ͗I(h)͘|]/[⌺ H ⌺ I I(h)]} Whermentioning

confidence: 99%

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

Leiros

Skagseth

Edvardsen

et al. 2014

“…The Asn208Arg mutation, which we identified as potentially being involved in protein dynamics, is also unlikely to play a major role because it would probably have affected the hydrolysis of the various substrates more uniformly. The remaining 3 amino acid differences between IMP-28 and IMP-1 are related to the L3 loop, which defines one side of the MBL active site and is known to be important for efficient hydrolysis (13). The Val223Ile and Leu241Ile differences found at both ends of this loop are not specific to the IMP-28 -IMP-1 pair, unlike the His306Gln mutation.…”

mentioning

confidence: 99%

Characterization of a Novel IMP-28 Metallo-β-Lactamase from a Spanish Klebsiella oxytoca Clinical Isolate

Pérez-Llarena

Fernández

Zamorano

et al. 2012

An isolate of Klebsiella oxytoca carrying a novel IMP metallo-␤-lactamase was discovered in Madrid, Spain. The bla IMP-28 gene is part of a chromosomally located class I integron. The IMP-28 k cat /K m values for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior.T he emergence of metallo-␤-lactamases (MBL) in members of the family Enterobacteriaceae is a problem of major concern for clinicians worldwide (11). These enzymes can hydrolyze most ␤-lactams, including carbapenems, and are not susceptible to conventional ␤-lactamase inhibitors (2).The IMP family has at least 33 unique IMP variants (http: //www.lahey.org/Studies), which may differ widely in regard to the primary sequence and biochemical activity. However, some allelic variants with the following mutations are associated with decreased overall activity (particularly against penicillins), i.e., Ser62 in IMP-12 (3), Ser196 in IMP-3 (6) and , and Gly242 in IMP-18 (1).Here we describe the genetic context and kinetic parameters of the new MBL IMP-28, which was first described in a Klebsiella oxytoca isolate from Spain, and in addition, we consider the possible cause of its poor overall activity.K. oxytoca HGUGM21530 was isolated from a lip wound patient seropositive for human immunodeficiency virus diagnosed with progressive multifocal leukoencephalopathy in the Gregorio Marañon Hospital (Madrid, Spain) in 2009.Pulsed-field gel electrophoresis (PFGE) with S1 nuclease digestion of whole-genome DNA (S1-PFGE) and PCR-based replicon typing (PBRT) were used to characterize plasmids as described previously (4). The S1-PFGE-I gel was transferred and hybridized with IMP and Inc A/C probes (the only amplicon obtained by PBRT). The results showed one band of 340 kb that hybridized only with the A/C probe. PFGE with I-CeuI digestion of wholegenome DNA, as described by Liu et al. (9), was used to determine whether the bla IMP-28 gene was located in the chromosome. The PFGE-I-CeuI gel was transferred and hybridized with 16S rRNA and IMP probes. The results showed one band that hybridized with both the 16S rRNA and IMP probes. These data suggest that the bla IMP-28 gene is located in the chromosome (data not shown).The genetic context of the bla IMP-28 gene was elucidated by PCR and sequencing. The bla gene is located in a class I integron, designated In767 (http://integrall.bio.ua.pt/), that displays more structural differences from (1,5,7,14,15,17) than similarities to (12, 18) the other integrons published for IMPencoding genes in the last decade. The structure consists of two aminoglycoside resistance genes, aacA44 and aadA13, just downstream of bla . The aacA44 gene codes for a newly described aminoglycoside-(6=)-acetyltransferase variant showing 86% sequence identity with AacA4.The bla IMP-1 and bla IMP-28 genes were cloned into...

“…Residue 228 has been thoroughly studied in several MBL enzymes; however, a proline variant similar to that found in TMB-2 has been described only in a VIM-2 R228P mutant (11). Residue 228 is located in MBL loop L3 (residues 223 to 240) and has been reported to contribute to substrate specificity (25,26) and to be involved in inhibitor binding (8,27).…”

mentioning

confidence: 99%

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

Skagseth

Christopeit

Akhter

et al. 2017

Metallo-␤-lactamases (MBLs) threaten the effectiveness of ␤-lactam antibiotics, including carbapenems, and are a concern for global public health. ␤-Lactam/␤-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-␤-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC 50 ) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC 50 levels of the new thiol-based inhibitors were 0.66 M (inhibitor 2a) and 0.62 M (inhibitor 2b), and the equilibrium dissociation constant (K D ) of inhibitor 2a was 1.6 M; thus, both were more potent inhibitors than L-captopril (IC 50 ϭ 47 M; K D ϭ 25 M). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped -stacking and R224 cation-interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than L-captopril.