Studies on the biosynthesis of riboflavin. 2. Further observations on nitrogen metabolism and flavinogenesis in Eremothecium ashbyii

Abstract

4,5-Diamino-2,6-dihydroxy-, 4,5-diamino-2-hydroxy- and 4,5-diamino-2,6-dihydroxy-pyrimidine, at a level of 8 mg N/100 ml, have little effect on either growth or flavinogenesis by E. ashbyii. 4,5,6-Triaminopyrimidine (I) inhibits growth and riboflavin synthesis equally (to about 50-60%), whilst 4,5,6-triamino-2-hydroxypyrimidine (n) inhibits riboflavin synthesis by about 65% but has little effect on growth. Compound (I) also inhibits riboflavin stimulation by adenine and xanthine and (II) that by xanthine only. Concentrations of aminopterin up to 2 [mu]g/100 ml inhibit growth but stimulate flavinogenesis considerably. Higher concentrations inhibit growth and flavinogenesis equally. Stimulation of flavinogenesis by adenine and threonine was not affected by 20 [mu]g/100 ml aminopterin but is somewhat inhibited by a level of 200 [mu]g/100 ml. Folinic acid (leucovorin) is more effective than folic acid in overcoming the growth inhibition of aminopterin. Azaxanthine inhibits riboflavin synthesis but not growth in E. ashbyii. Riboflavin stimulation by adenine is inhibited to a greater extent than that by xanthine. Alloxan and 4-amino-l,2-dimethyl-5-(D-l''-ribitylamino) benzene together do not stimulate flavinogenesis, although they react spontaneously in the culture medium to form, not riboflavin, but what is probably the related anil. 2-amino-and 2-hydroxy-purine do not stimulate riboflavin synthesis markedly, whilst 2-methyladenine and 2-methylxanthine have a somewhat greater effect. None of these affects growth, but the first two inhibit the stimulation of flavinogenesis by threonine. Pyruvate was the only non -nitrogenous compound related to serine or threonine which stimulated riboflavin synthesis. Etnanolamine, but not glycine - formate, was also active. E. ashbyii does not grow on acetate alone and, in the presence of glucose, acetate considerably inhibits riboflavin synthesis. Washed colorless cells of E. ashbyii produced by culturing in the absence of glucose synthesize small amounts of riboflavin.