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

Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

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.
Originalspracheenglisch
Seiten (von - bis)25411-25426
Seitenumfang16
FachzeitschriftThe Journal of Biological Chemistry
Jahrgang291
Ausgabenummer49
DOIs
PublikationsstatusVeröffentlicht - 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
Electrons
Ligands

Schlagwörter

  • E. coli
  • enzyme kinetics
  • nitrile reductase

ASJC Scopus subject areas

  • !!Biochemistry
  • !!Molecular Biology
  • Zellbiologie

Dies zitieren

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, Jahrgang 291, Nr. 49, 2016, S. 25411-25426.

Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

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keywords = "E. coli, enzyme kinetics, nitrile reductase",
author = "Jihye Jung and Tibor Czabany and Birgit Wilding and Norbert Klempier and Bernd Nidetzky",
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T1 - Kinetic analysis and probing with substrate analogues of the reaction pathway of the nitrile reductase QueF from Escherichia coli

AU - Jung, Jihye

AU - Czabany, Tibor

AU - Wilding, Birgit

AU - Klempier, Norbert

AU - Nidetzky, Bernd

PY - 2016

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N2 - 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.

AB - 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.

KW - E. coli

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