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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


The nitrile reductase QueF catalyzes NADPH-dependent reduction of the nitrile group of preQ0 (7-cyano-7-deazaguanine) into the primary amine of preQ1 (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 kcat≈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.

Original languageEnglish
Pages (from-to)1534-1543
Number of pages10
Issue number10
Publication statusPublished - 15 May 2020


  • C=C double bond hydration
  • cofactors
  • hydration
  • nitrile reductases

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

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