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Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli
Author(s) -
Jung Jihye,
Braun Jan,
Czabany Tibor,
Nidetzky Bernd
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900679
Subject(s) - chemistry , nitrile hydratase , nitrile , stereochemistry , moiety , escherichia coli , protonation , double bond , amine gas treating , reductase , enzyme , biochemistry , organic chemistry , ion , gene
The nitrile reductase QueF catalyzes NADPH‐dependent reduction of the nitrile group of preQ 0 (7‐cyano‐7‐deazaguanine) into the primary amine of preQ 1 (7‐aminomethyl‐7‐deazaguanine), a biologically unique reaction important in bacterial nucleoside biosynthesis. Here we have discovered that the QueF from Escherichia coli —its D197A and E89L variants in particular (apparent k cat ≈10 −2 min −1 )—also catalyze the slow hydration of the C5=C6 double bond of the dihydronicotinamide moiety of NADPH. The enzymatically C6‐hydrated NADPH is a 3.5:1 mixture of R and S forms and rearranges spontaneously through anomeric epimerization (β→α) and cyclization at the tetrahydronicotinamide C6 and the ribosyl O2. NADH and 1‐methyl‐ or 1‐benzyl‐1,4‐dihydronicotinamide are not substrates of the enzymatic hydration. Mutagenesis results support a QueF hydratase mechanism, in which Cys190—the essential catalytic nucleophile for nitrile reduction—acts as the general acid for protonation at the dihydronicotinamide C5 of NADPH. Thus, the NADPH hydration in the presence of QueF bears mechanistic resemblance to the C=C double bond hydration in natural hydratases.