title: Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli.
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 preQ0and a cysteine nucleophile in the enzyme, and this adduct is subsequently converted into preQ1in two NADPH-dependent reduction steps. Here, we show that the Escherichia coli QueF binds preQ0in 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 preQ0reduction involve transfer of the 4-pro-R-hydrogen from NADPH. They proceed about 4-7-fold more slowly than trapping of the enzyme-bound preQ0as 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 preQ17-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.
authors.affiliation: From the Austrian Centre of Industrial Biotechnology, Petersgasse 14.|the Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWIGraz, Petersgasse 12/1, and.
authors.forename: Jihye
authors.initials: J
authors.lastname: Jung
authors.affiliation: the Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWIGraz, Petersgasse 12/1, and.
authors.forename: Tibor
authors.initials: T
authors.lastname: Czabany
authors.affiliation: From the Austrian Centre of Industrial Biotechnology, Petersgasse 14.|the Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria.
authors.forename: Birgit
authors.initials: B
authors.lastname: Wilding
authors.affiliation: the Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria.
authors.forename: Norbert
authors.initials: N
authors.lastname: Klempier
authors.affiliation: From the Austrian Centre of Industrial Biotechnology, Petersgasse 14, bernd.nidetzky@tugraz.at.|the Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWIGraz, Petersgasse 12/1, and.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000737:chemistry*; D009704:Nucleoside Q / Q000031:analogs & derivatives* / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*
chemical_list: C556329:7-cyano-7-deazaguanosine; D029968:Escherichia coli Proteins; D006151:Guanosine; D009249:NADP; D009704:Nucleoside Q; C021610:7-aminomethyl-7-deazaguanosine; D010088:Oxidoreductases; C582776:QueF protein, E coli
keywords: NADPH; catalytic reaction pathway; enzyme catalysis; enzyme mechanism; isothermal titration calorimetry (ITC); isotope effect; nitrile reductase; pre-steady-state kinetics; reductase; thermodynamics