Interplay of nucleophilic catalysis with proton transfer in the nitrile reductase QueF from <i>Escherichia coli</i>

Jung, Jihye; Braun, Jan; Czabany, Tibor; Nidetzky, Bernd

doi:10.1039/c8cy02331j

Cited by 3 publications

(6 citation statements)

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“…Single‐site variants of ecQueF (C190A, C190S, D197A, D197H) were isolated and characterized for activity in preQ 0 reduction, along with the wild‐type enzyme . Catalytic reactions of the variants with preQ 0 were peculiar in that their NADPH consumption greatly exceeded the corresponding preQ 1 release, and the excess utilization of NADPH was largely uncoupled from the formation of NADP + .…”

Section: Resultsmentioning

confidence: 99%

“…The proposed mechanism of QueF catalysis involves a thioimidate covalent enzyme‐preQ 0 adduct that undergoes reduction by NADPH in two catalytic steps via an imine intermediate (Scheme ) . The QueF active site consists of a cysteine/aspartate dyad of residues that operate in a functionally interconnected manner in covalent enzyme catalysis, as shown in Scheme . As part of our investigation of the QueF mechanism, we substituted the relevant Cys190 (by Ala and Ser) and Asp197 (by Ala and His) in the enzyme from Escherichia coli (ecQueF) and studied preQ 0 reduction in the presence of the enzyme variants .…”

Section: Methodsmentioning

confidence: 99%

“…The QueF active site consists of a cysteine/aspartate dyad of residues that operate in a functionally interconnected manner in covalent enzyme catalysis, as shown in Scheme . As part of our investigation of the QueF mechanism, we substituted the relevant Cys190 (by Ala and Ser) and Asp197 (by Ala and His) in the enzyme from Escherichia coli (ecQueF) and studied preQ 0 reduction in the presence of the enzyme variants . For certain enzyme variants (e.g., D197A) we noted that a large amount of the NADPH consumed was not accounted for either in the preQ 0 converted or in the NADP + released.…”

Section: Methodsmentioning

confidence: 99%

“…Site‐directed mutagenesis and enzyme preparation : Mutagenesis to substitute Glu89 with Leu (E89L), Cys190 with Ala (C190A) and Ser (C190S), and Asp197 with Ala (D197A) or His (D197H) was reported in earlier studies of ecQueF . Genes encoding the double variants (C190A/D197A, E89L/D197A, E89Q/D197A) were obtained from Genscript (Piscataway, NJ, USA).…”

Section: Methodsmentioning

confidence: 99%

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Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

et al. 2020

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Scheme1.Proposed mechanism of reduction of preQ 0 into preQ 1 ,catalyzed by the nitrile reductase QueF.The active-site dyadCys and Asp act together in covalentcatalysis. Reduction of the thioimidate enzyme adduct occurs in two NADPH-dependent stepsv ia an iminei ntermediate (not shown).Scheme2.Reactionpathways leadingtoN ADPH degradation in spontaneous and enzyme-promoted conversions.The uncoupled pathway (NADPH conversion not leading to NADP + )i nvolves hydration of NADPH to NADPHX followed by epimerization/cyclization, leading to O2'-b-6-cyclo-1,4,5,6-tetrahydronicotinamideadenine dinucleotidephosphate [(cTHN)TPN]. The enzymatic reaction identified in this study is markedb yt he box. The coupled pathway (NADPH conversion leadingt oN ADP + )entailsformation of NADP + through knowno xidationr eactionso fN ADPH: whenO 2 is present, for example.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

et al. 2020

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“…[23][24][25][26] Many biological processes like enzyme catalysis rely on nucleophiles formed by proton transfer reactions. [27][28][29] For example, the self-cleavage mechanism in hairpin ribosomes proceeds via proton transfer involving internal nucleophiles. 30 Moreover, Neves et al 19 reported nucleophilic disulfide bond rupture by the proton transfer mechanism in Protein Disulfide Isomerase (PDI), the mammalian counterpart of nDsbD.…”

Section: Introductionmentioning

confidence: 99%

Towards solvent regulated self-activation of N-terminal disulfide bond oxidoreductase-D

Nair

Perumalla

Anjukandi

2022

Phys. Chem. Chem. Phys.

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QM/MM Studies on Enzyme Catalysis and Insight into Designing of New Inhibitors by ONIOM Approach: Recent Update

et al. 2023

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Computational enzymology is a rapidly developing area that uniquely provides deep insight into the fundamental processes of biological catalysis at the atomic level. Such in‐depth insight can ultimately be employed in designing potential inhibitors against the targets of interest. Computational enzymology covers a wide range of in‐silico approaches for investigating the enzyme‐catalyzed reaction mechanisms, among which combined quantum mechanics (QM) /molecular mechanics (MM) approaches have gained a lot of attention nowadays. This advanced approach generally involves a QM method (i. e. a method that estimates the electronic structure of the active site) and a simpler MM method (a method that includes the enzyme environment) to understand the enzymatic reactions. The QM/MM method has been widely tested in understanding the molecular mechanisms both at the structural and energetic levels and observed to best correlate with experimental studies of the enzymatic mechanism. It proposes a new mechanism that ultimately opens a new route for designing new potent, efficacious enzyme inhibitors. This review mainly covers wide applications of the ONIOM (Our own N‐layer Integrated molecular Orbital Molecular mechanics) method for decoding the enzymatic catalysis mechanism or designing potential small molecule inhibitors as treatment therapeutics in terms of free energy profiles. Moreover, this article also highlights employing QM/MM method in comprehending the mechanisms for drug metabolism and resistance (owing to mutations). This write‐up may encourage medicinal chemists and molecular biologists to explore this approach to propose more promising therapeutics to improve the quality of treatment.

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Interplay of nucleophilic catalysis with proton transfer in the nitrile reductase QueF from Escherichia coli

Abstract: Proton relay through an active-site network of hydrogen bonds promotes enzymatic nitrile reduction to amine via a covalent thioimidate enzyme intermediate.

Cited by 3 publications

References 49 publications

Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

Towards solvent regulated self-activation of N-terminal disulfide bond oxidoreductase-D

QM/MM Studies on Enzyme Catalysis and Insight into Designing of New Inhibitors by ONIOM Approach: Recent Update

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