Expression in Escherichia coli of an unnamed protein gene from Aspergillus oryzae RIB40 and cofactor analyses of the gene product as formate oxidase.
An unnamed protein of Aspergillus oryzae RIB40 (accession no. XP_001727378), the amino acid sequence of which shows high similarity to those of formate oxidase isoforms produced by Debaryomyces vanjiriae MH201, was produced in Escherichia coli in C-His(6)-tagged form. The gene product, purified by affinity column chromatography, catalyzed the oxidation of formate to yield hydrogen peroxide but showed no evidence of activity on the other substrates tested. The K(m) and V(max) values at 30 degrees C at pH 4.5 were 7.9 mM and 26.3 micromole/min mg respectively. The purified enzyme showed UV-visible spectra atypical of ordinary flavoproteins. The UV-visible spectra of the enzyme and the UV-visible spectra, fluorescence spectra, and mass spectrometry of the extract obtained by boiling the purified enzyme suggested that the enzyme has a non-covalently bound FAD analog, which is expected to be 8-formyl-FAD.
Maeda
Yoshifumi
Y
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, Japan.
Doubayashi
Daiju
D
Oki
Masaya
M
Nose
Hiroaki
H
Sakurai
Akihiko
A
Isa
Kimio
K
Fujii
Yutaka
Y
Uchida
Hiroyuki
H
D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D055162:Biocatalysis; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D005561:Formates / Q000032:analysis / Q000378:metabolism*; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D055550:Protein Stability; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013696:Temperature
D003067:Coenzymes; D005561:Formates; D005182:Flavin-Adenine Dinucleotide; D006861:Hydrogen Peroxide; D010088:Oxidoreductases