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

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 Qu...

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Detalles Bibliográficos
Autores principales: Jung, Jihye, Braun, Jan, Czabany, Tibor, Nidetzky, Bernd
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317782/
https://www.ncbi.nlm.nih.gov/pubmed/31850614
http://dx.doi.org/10.1002/cbic.201900679
Descripción
Sumario: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.