title: C4a-hydroperoxyflavin formation in N-hydroxylating flavin monooxygenases is mediated by the 2'-OH of the nicotinamide ribose of NADP⁺.
abstract: Flavin-dependent monooxygenases must stabilize a C4a-hydroperoxyflavin intermediate to hydroxylate their respective substrates. Formation and decay of the C4a-hydroperoxyflavin were monitored under rapid reaction kinetic conditions in SidA, an N-hydroxylating monooxygenase involved in siderophore biosynthesis. Solvent kinetic isotope effect studies of flavin oxidation indicate that both hydrogen peroxide elimination and water elimination occur via abstraction of hydrogen from the N5 of the flavin. Kinetic isotope effect and density functional theory results are consistent with the transfer of a proton from the 2'-OH of the nicotinamide ribose of nicotinamide adenine dinucleotide phosphate (NADP⁺) to the C4a-peroxyflavin to form the C4a-hydroperoxyflavin. This represents a novel role for NADP⁺ in the reaction of flavin-dependent enzymes.
authors: Department of Biochemistry and ‡Virginia Tech Center for Drug Discovery, Virginia Tech , Blacksburg, Virginia 24061, United States.
authors: Reeder
authors: R
authors: Robinson
authors: Somayesadat
authors: S
authors: Badieyan
authors: Pablo
authors: P
authors: Sobrado
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology; D055162:Biocatalysis; D019295:Computational Biology / Q000379:methods; D041961:Deuterium Exchange Measurement; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D009952:Ornithine / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D011522:Protons
chemical_list: C531259:C4a-hydroperoxyflavin; D005415:Flavins; D005656:Fungal Proteins; D011522:Protons; D009249:NADP; D009952:Ornithine; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)