Dataset Information

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

ABSTRACT: Most ene-reductases belong to the Old Yellow Enzyme (OYE) family of flavin-dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron-withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X-ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual-specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a ?-hairpin motif in an active site loop of both these enzymes were studied using stopped-flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the ?-hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15-fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55-fold) increase in efficiency in the reductive half-reaction, when compared to the essentially unreactive wild-type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs.

SUBMITTER: Iorgu AI

PROVIDER: S-EPMC6767020 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Publications

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

Iorgu Andreea I AI Hedison Tobias M TM Hay Sam S Scrutton Nigel S NS

The FEBS journal 20190513 16

Most ene-reductases belong to the Old Yellow Enzyme (OYE) family of flavin-dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron-withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X-ray crystallographic data were used to rationally design variants of two OYEs, penta ...[more]

PMID: 31033202

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Dataset Information

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

Publications

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

Similar Datasets

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