Kinetic analysis and probing with substrate analogues of the reaction pathway of the nitrile reductase QueF from Escherichia coli

Jihye Jung, Tibor Czabany, Birgit Wilding, Norbert Klempier, Bernd Nidetzky

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The enzyme QueF catalyzes a four-electron reduction of a nitrile group into an amine, the only reaction of this kind known in biology. In nature, QueF converts 7-cyano-7-deazaguanine (preQ0) into 7-aminomethyl-7-deazaguanine (preQ1) for the biosynthesis of the tRNA-inserted nucleoside queuosine. The proposed QueF mechanism involves a covalent thioimide adduct between preQ0 and a cysteine nucleophile in the enzyme, and this adduct is subsequently converted into preQ1 in two NADPH-dependent reduction steps. Here, we show that the Escherichia coli QueF binds preQ0 in a strongly exothermic process (ΔH = −80.3 kJ/mol; −TΔS = 37.9 kJ/mol, Kd = 39 nM) whereby the thioimide adduct is formed with half-of-the-sites reactivity in the homodimeric enzyme. Both steps of preQ0 reduction involve transfer of the 4-pro-R-hydrogen from NADPH. They proceed about 4–7-fold more slowly than trapping of the enzyme-bound preQ0 as covalent thioimide (1.63 s−1) and are thus mainly rate-limiting for the enzyme's kcat (=0.12 s−1). Kinetic studies combined with simulation reveal a large primary deuterium kinetic isotope effect of 3.3 on the covalent thioimide reduction and a smaller kinetic isotope effect of 1.8 on the imine reduction to preQ1. 7-Formyl-7-deazaguanine, a carbonyl analogue of the imine intermediate, was synthesized chemically and is shown to be recognized by QueF as weak ligand for binding (ΔH = −2.3 kJ/mol; −TΔS = −19.5 kJ/mol) but not as substrate for reduction or oxidation. A model of QueF substrate recognition and a catalytic pathway for the enzyme are proposed based on these data.
LanguageEnglish
Pages25411-25426
Number of pages16
JournalThe Journal of Biological Chemistry
Volume291
Issue number49
DOIs
StatusPublished - 2016

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Nitriles
Escherichia coli
Oxidoreductases
Kinetics
Substrates
Enzymes
Imines
NADP
Isotopes
Nucleoside Q
Nucleophiles
Deuterium
Biosynthesis
Transfer RNA
Nucleosides
Amines
Cysteine
Hydrogen
Cats
Electrons

Keywords

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    Kinetic analysis and probing with substrate analogues of the reaction pathway of the nitrile reductase QueF from Escherichia coli. / Jung, Jihye; Czabany, Tibor; Wilding, Birgit; Klempier, Norbert; Nidetzky, Bernd.

    In: The Journal of Biological Chemistry, Vol. 291, No. 49, 2016, p. 25411-25426.

    Research output: Contribution to journalArticleResearchpeer-review

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