Occurrence of a vitamin B2-aldehyde-forming enzyme in Schizophyllum commune.

Vitamin B2-aldehyde-forming enzyme from riboflavin was purified about 1,400-fold from a cell-free extract of Schizophyllum commune by ammonium sulfate saturation fractionation, ethanol fractionation, and column chromatographies on DEAE-Sephacel and Sephadex G-100. The purified enzyme was shown to be homogeneous on polyacrylamide disc gel electrophoresis, and most active at about pH 5.5. The molecular weight was determined to be approximately 60,000-62,000 by gel filtration. The enzyme was specific for riboflavin; other compounds such as alcohol, sugar, phenol or nucleoside were not oxidized by this enzyme, as far as tested. alpha-NAD+, beta-NAD+, alpha-NADP+, beta-NADP+, FMN, FAD, and cytochrome c were not active as an electron acceptor. 2,6-Dichlorophenolindophenol served as a good electron acceptor, and phenazine methosulfate and methylene blue were found to be somewhat effective. The enzyme stoichiometrically oxidized 1 mol of riboflavin with 1 mol of 2,6-dichlorophenolindophenol as electron acceptor. The enzyme reaction was completely inhibited by 10 microM Hg2+, but was not inhibited by sulfhydryl reagent, carbonyl reagent, and metal chelator. Kinetics analysis gave a Km value of 17 microM for riboflavin.