ABSTRACT
Formate oxidase of Aspergillus oryzae RIB40 contains an 8-replaced FAD with molecular mass of 799 as cofactor. The ¹H-NMR spectrum of the cofactor fraction obtained from the enzyme indicated that the 8-replaced FAD in the fraction was 8-formyl-FAD, present in open form and hemiacetal form. The oxidation-reduction potentials of the open and hemiacetal forms were estimated by cyclic voltammetry to be -47 and -177 mV vs. Normal Hydrogen Electrode respectively. The structure of the enzyme was constructed using diffraction data to 2.24 Å resolution collected from a crystal of the enzyme. His511 and Arg554 were situated close to the pyrimidine part of the isoalloxazine ring of 8-formyl-FAD in open form. The enzyme had 8-formyl-FAD, the oxidation potential of which was approximately 160 mV more positive than that of FAD, and the His-Arg pair at the catalytic site, unlike the other enzymes belonging to the glucose-methanol-choline oxidoreductase family.
Subject(s)
Alcohol Oxidoreductases/metabolism , Aspergillus oryzae/enzymology , Flavin-Adenine Dinucleotide/metabolism , Fungal Proteins/metabolism , Recombinant Proteins/metabolism , Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/genetics , Aspergillus oryzae/chemistry , Binding Sites , Catalytic Domain , Choline/metabolism , Crystallography, X-Ray , Escherichia coli , Flavin-Adenine Dinucleotide/chemistry , Formates/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Magnetic Resonance Spectroscopy , Methanol/metabolism , Models, Molecular , Oxidation-Reduction , Potentiometry , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Formate oxidase (FOD), which catalyzes the oxidation of formate to yield carbon dioxide and hydrogen peroxide, belongs to the glucose-methanol-choline oxidoreductase (GMCO) family. FOD from Aspergillus oryzae RIB40, which has a modified FAD as a cofactor, was crystallized at 293 K by the hanging-drop vapour-diffusion method. The crystal was orthorhombic and belonged to space group C222(1). Diffraction data were collected from a single crystal to 2.4 A resolution.
