Metabolism of riboflavin in Schizophyllum commune.

Abstract

Two new flavin compounds (X and Y) were found as metabolites of Schizophyllum commune, a Basidiomycete, producing a larger amount of L-malate through CO2-fixing process such as reductive carboxylation of phosphoenolpyruvate coupled with glyceraldehyde-3-phosphate under aerobic conditions. These two flavins were named ‘Schizoflavin (SF)’ after Schizophyllum, and X and Y were tentatively called SF1 and SF2, respectively. By using an enzymic system, SF2 was demonstrated to be a direct precursor of SF1, being formed from riboflavin. Experiments were designed to obtain information about the relationship between SF and L-malate production in the mold. The conversion yield of SF from riboflavin was found to correspond to the level of L-malate-producing activity of the strains under aerobic conditions, where a stimulation of L-malate accumulation by SF1 was observed. Enzymic studies of mycelial homogenate of the mold showed. that SF1 did not stimulate the degradation of L-malate, although FAD did markedly so to form pyruvate. SF was found not only to be distributed in all strains of S. commune, so far as tested, but also to be present in edible Basidiomycetes containing Agaricus bisporus and some uncharacterized Basidiomycetes. SF1 was isolated, crystallized and identified as 7,8-dimethyl-10-(2,3,4-trihydroxy-4-carboxybutyl)isoalloxazine, and SF2 was identified as 7,8-dimethyl-10-(2,3,4-trihydroxy-4-formylbutyl)isoalloxazine. SF1 and SF2 were designated vitamin B2-acid and vitamin B2-aldehyde, respectively. The vitamin activity of B2-aldehyde was proved by the bioassay using Lactobacillus casei. Reduction of B2-aldehyde to riboflavin was demonstrated by the cell-free extract of L. casei. Vitamin B2-aldehyde-forming enzyme was isolated and shown so far to be a novel enzyme which is specific for riboflavin. α-NAD+, β-NAD+, α-NADP+, β-NADP+, FMN, FAD, and cytochrome c do not serve as electron acceptors for the enzyme, whereas 2,6-dichlorophenolindophenol, phenazine methosulfate, and methylene blue do so. However, NADPH-dependence of the enzyme and O2 requirement have been elucidated in the presence of Cu2+. The enzyme reaction was inhibited by α-tocopherol. It was suggested that the over-production of L-malate through a reductive CO2-fixing process under aerobic conditions was due to FAD deficiency caused by vitamin B2-acid and vitamin B2-aldehyde formation because of the depression of pyruvate catabolism and L-malate degradation that require FAD, and due to the activity of vitamin B2-acid itself.