Metabolism of riboflavin in Schizophyllum commune

Summary

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.