The NAD(P)H:flavin oxidoreductase from Escherichia coli. Evidence for a new mode of binding for reduced pyridine nucleotides.
The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or NADH as electron donor. The enzyme does not contain any prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin in a ternary complex prior to oxidoreduction. The specificity of the flavin reductase for the pyridine nucleotide was studied by steady-state kinetics using a variety of NADP analogs. Both the nicotinamide ring and the adenosine part of the substrate molecule have been found to be important for binding to the polypeptide chain. However, in the case of NADPH, the 2'-phosphate group destabilized almost completely the interaction with the adenosine moiety. Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and we have shown that both these molecules bind to the enzyme almost exclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine nucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore classified as an A-side-specific enzyme.
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Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DBMS-CEA/CNRS/Université J. Fourier, Batiment K, 17 Avenue des Martyrs, 38054 Grenoble, Cedex 9, France. niviere@cbcrb.ceng.cea.fr
Fieschi
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Dećout
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JL
Fontecave
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D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D038181:FMN Reductase; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D013237:Stereoisomerism
D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases