Crystal structures of VIM‐1 complexes explain active site heterogeneity in VIM‐class metallo‐β‐lactamases

Salimraj, Ramya; Hinchliffe, P.; Kosmopoulou, Magda; Tyrrell, Jonathan M.; Brem, Jürgen; Berkel, Sander S. van; Verma, Anil Kumar; Owens, Raymond J.; McDonough, M.A.; Walsh, Timothy R.; Schofield, Christopher J.; Spencer, James

doi:10.1111/febs.14695

Cited by 31 publications

(47 citation statements)

References 62 publications

(104 reference statements)

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“…The tyrosine-to-histidine substitution is located 18 Å, a C ␣ -C ␣ distance, from the H229R change, and in silico mutation of VIM-20 Tyr201 to histidine does not suggest any perturbation to the position of Arg229. In comparison, VIM-59 is a VIM-1-like variant; thus, modeling of the H229R substitution required using VIM-1 as the starting point (PDB ID 5N5G) (24). After introducing the H229R change in silico in the VIM-1 background, we found that H229R was easily accommodated (Fig.…”

Section: Structural Characterization Of Vim-20mentioning

confidence: 98%

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A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions

Cheng

Bethel

Thomas³

et al. 2019

mBio

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To understand the evolution of Verona integron-encoded metallo-β-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-β-lactamase families, although compensating mutations use different mechanisms to enhance resistance. IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-β-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-β-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.

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Section: Structural Characterization Of Vim-20mentioning

confidence: 98%

“…In silico substitutions. Comparative models for VIM-1 and VIM-59 began using the holo structure of reduced VIM-1 (PDB ID 5N5G) (24). A model for VIM-59 was prepared by introducing the H229R mutation in silico using Coot (45).…”

Section: Nano-esi-ms-based Analysismentioning

confidence: 99%

A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions

Cheng

Bethel

Thomas³

et al. 2019

mBio

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“…In addition to substrate-metal interactions, several works have shown that active site L3 and L10 in B1 enzymes play an important role in substrate binding [ 60 , 132 , 133 , 134 , 135 , 136 , 137 ]. Based on these experimental results, in the availability of different crystal structures of EP complexes [ 69 , 101 , 135 , 138 , 139 , 140 ] and many docking studies [ 49 , 105 , 106 , 141 , 142 ], a general substrate binding mode has been proposed.…”

Section: Mbl Inhibitors Based On Substrate Structuresmentioning

confidence: 99%

“…Further interactions are the C7 carboxylate with Asn233 and the C3 carboxylate with Asn233, Lys224, and Val67. Enzymes from the VIM family lack Lys224, and the carboxylate oxygen bound to Zn2 interacts with an extra water molecule and backbone atoms from Cys221 [ 140 ]. The different substituents are located on the grooves defined by the active site L3 and L10, but few specific interactions have been reported [ 101 , 134 , 139 ].…”

Section: Intermediate Species and Enzyme: Product Complexes As Temmentioning

confidence: 99%

Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism

et al. 2020

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β-Lactam antibiotics are the most widely prescribed antibacterial drugs due to their low toxicity and broad spectrum. Their action is counteracted by different resistance mechanisms developed by bacteria. Among them, the most common strategy is the expression of β-lactamases, enzymes that hydrolyze the amide bond present in all β-lactam compounds. There are several inhibitors against serine-β-lactamases (SBLs). Metallo-β-lactamases (MBLs) are Zn(II)-dependent enzymes able to hydrolyze most β-lactam antibiotics, and no clinically useful inhibitors against them have yet been approved. Despite their large structural diversity, MBLs have a common catalytic mechanism with similar reaction species. Here, we describe a number of MBL inhibitors that mimic different species formed during the hydrolysis process: substrate, transition state, intermediate, or product. Recent advances in the development of boron-based and thiol-based inhibitors are discussed in the light of the mechanism of MBLs. We also discuss the use of chelators as a possible strategy, since Zn(II) ions are essential for substrate binding and catalysis.

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“…VIM-1. The available structures of VIM-1 co-crystallized with hydrolyzed meropenem was selected as the target structure (PDB code 5n5i) [59]. The docking simulations were guided by the recently solved crystallographic structure of one thio-triazole derivative complexed with VIM-2 (PDB code 6tgi).…”

Section: Static Dockingmentioning

confidence: 99%

4-Amino-1,2,4-triazole-3-thione as a Promising Scaffold for the Inhibition of Serine and Metallo-β-Lactamases

et al. 2020

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The emergence of bacteria that co-express serine- and metallo- carbapenemases is a threat to the efficacy of the available β-lactam antibiotic armamentarium. The 4-amino-1,2,4-triazole-3-thione scaffold has been selected as the starting chemical moiety in the design of a small library of β-Lactamase inhibitors (BLIs) with extended activity profiles. The synthesised compounds have been validated in vitro against class A serine β−Lactamase (SBLs) KPC-2 and class B1 metallo β−Lactamases (MBLs) VIM-1 and IMP-1. Of the synthesised derivatives, four compounds showed cross-class micromolar inhibition potency and therefore underwent in silico analyses to elucidate their binding mode within the catalytic pockets of serine- and metallo-BLs. Moreover, several members of the synthesised library have been evaluated, in combination with meropenem (MEM), against clinical strains that overexpress BLs for their ability to synergise carbapenems.

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Crystal structures of VIM‐1 complexes explain active site heterogeneity in VIM‐class metallo‐β‐lactamases

Cited by 31 publications

References 62 publications

A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions

A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions

Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism

4-Amino-1,2,4-triazole-3-thione as a Promising Scaffold for the Inhibition of Serine and Metallo-β-Lactamases

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