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Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: species comparison and structural changes with substrate binding and release.

ABSTRACT: NAD(P)H/quinone acceptor oxidoreductase (QR1, NQO1, formerly DT-diaphorase; EC ) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. In this paper we report the apoenzyme structures of human (at 1.7-A resolution) and mouse (2.8 A) QR1 and the complex of the human enzyme with the substrate duroquinone (2.5 A) (2,3,5, 6-tetramethyl-p-benzoquinone). In addition to providing a description and rationale of the structural and catalytic differences among several species, these structures reveal the changes that accompany substrate or cofactor (NAD) binding and release. Tyrosine-128 and the loop spanning residues 232-236 close the binding site, partially occupying the space left vacant by the departing molecule (substrate or cofactor). These changes highlight the exquisite control of access to the catalytic site that is required by the ping-pong mechanism in which, after reducing the flavin, NAD(P)(+) leaves the catalytic site and allows substrate to bind at the vacated position. In the human QR1-duroquinone structure one ring carbon is significantly closer to the flavin N5, suggesting a direct hydride transfer to this atom.

SUBMITTER: Faig M

PROVIDER: S-EPMC16212 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature

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Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: species comparison and structural changes with substrate binding and release.

Faig M M Bianchet M A MA Talalay P P Chen S S Winski S S Ross D D Amzel L M LM

Proceedings of the National Academy of Sciences of the United States of America 20000301 7

NAD(P)H/quinone acceptor oxidoreductase (QR1, NQO1, formerly DT-diaphorase; EC ) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. In this paper we report the apoenzyme structures of human (at 1.7-A resolution) and mouse (2.8 A) QR1 and the complex of the human enzyme with the substrate duroquinone (2.5 A) (2,3,5, 6-tetramethyl-p-benzoquinone). In addition to providing a description and rationale of the structural and catalytic differences a ...[more]

PMID: 10706635

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Project description:BackgroundOxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose-methanol-choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily.ResultsIn this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold.ConclusionsThe phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions.

Project description:NAD(P)H:quinone oxidoreductase type I (NQO1) is a target enzyme for triggered delivery of drugs at inflamed tissue and tumor sites, particularly those that challenge traditional therapies. Prodrugs, macromolecules, and molecular assemblies possessing trigger groups that can be cleaved by environmental stimuli are vehicles with the potential to yield active drug only at prescribed sites. Furthermore, quinone propionic acids (QPAs) covalently attached to prodrugs or liposome surfaces can be removed by application of a reductive trigger stimulus, such as that from NQO1; their rates of reductive activation should be tunable via QPA structure. We explored in detail the recombinant human NAD(P)H:quinone oxidoreductase type I (rhNQO1)-catalyzed NADH reduction of a family of substituted QPAs and obtained high precision kinetic parameters. It is found that small changes in QPA structure-in particular, single atom and function group substitutions on the quinone ring at R(1)-lead to significant impacts on the Michaelis constant (K(m)), maximum velocity (V(max)), catalytic constant (k(cat)), and catalytic efficiency (k(cat)/K(m)). Molecular docking simulations demonstrate that alterations in QPA structure result in large changes in QPA alignment and placement with respect to the flavin isoalloxazine ring in the active site of rhNQO1; a qualitative relationship exists between the kinetic parameters and the depth of QPA penetration into the rhNQO1 active site. From a quantitative perspective, a very good correlation is observed between log(k(cat)/K(m)) and the molecular-docking-derived distance between the flavin hydride donor site and quinone hydride acceptor site in the QPAs, an observation that is in agreement with developing theories. The comprehensive kinetic and molecular modeling knowledge obtained for the interaction of recombinant human NQO1 with the quinone propionic acid analogues provides insight into the design and implementation of the QPA trigger groups for drug delivery applications.

Dataset Information

Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: species comparison and structural changes with substrate binding and release.

Publications

Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: species comparison and structural changes with substrate binding and release.

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