Wild-type and saccharopine dehydrogenaseless mutant strains of Rhodotorula glutinis grew in minimal medium containing lysine as the sole nitrogen source and simultaneously accumulated, in the culture supematant, large amounts of a product identified as a-aminoadipic-8-semialdehyde. The saccharopine dehydrogenase and pipecolic acid oxidase levels remained unchanged in wild-type cells grown in the presence of ammonium or lysine as the nitrogen source. Lysine-aketoglutarate aminotransferase activity was demonstrated in ammonium-grown cells. This activity was derepressed in cells grown in the presence of lysine as the sole source of nitrogen.

The role of pipecolic acid in the biosynthesis of lysine was investigated in Rhodotorula glutinis, an aerobic red yeast. Supplementation of pipecolic acid in the minimal medium supported the growth of mutants lys2, Iys3, and lys5; aaminoadipic acid supported the growth of lys5; but neither a-aminoadipic acid nor pipecolic acid supported the growth of mutants MNNG42 and MNNG37. During the growth of the appropriate mutants, pipecolic acid was removed from the growth medium and the intraceUular pool. In tracer experiments, radioactivity from ["4C]pipecolic acid was selectively incorporated into the cellular lysine of lys5 and the wild-type strain. L-Pipecolic acid-dependent enzyme activity did not require any cofactor and was inhibited by mercuric chloride and potassium cyanide. This activity was present in the wild-type strain and all of the mutants tested and was repressed in mutant lys5 when grown in the presence of higher concentration of lysine. The reaction product of pipecolic acid was converted to saccharopine by lysS enzyme in the presence of glutamate and reduced nicotinamide adenine dinucleotide phosphate. Mutant MNNG37 lacked the saccharopine dehydrogenase activity, indicating that this step is involved in the conversion of a-aminoadipic acid and pipecolic acid to lysine. Mutants MNNG37 and MNNG42 accumulated a p-dimethylaminobenzaldehyde-reacting product in the culture supernatant and in the intracellular pool. Chromatographic properties of the p-dimethylaminobenzaldehyde adduct and that of the pipecolic acid-dependent reaction product were similar. The reaction product and the accumulation product were characterized on the basis of mass and absorption spectra as aaminoadipic-semialdehyde, which in solution remains in equilibrium with A'piperideine-6-carboxylic acid. Since a-aminoadipic-semialdehyde is a known intermediate of the a-aminoadipic acid pathway for the biosynthesis of lysine, it is concluded that pipecolic acid is converted to lysine in R. glutinis via a-aminoadipic-semialdehyde and saccharopine.

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Pipecolic acid oxidase from Rhodotorula glutinis, which converts pipecolic acid to a-aminoadipic-&-semialdehyde, an intermediate of the biosynthetic pathway of lysine, was purified 290-fold. The enzyme from the crude extract and purified preparation exhibited a molecular weight of approximately 43,000 and was composed of a single subunit. The purified enzyme was heat labile and exhibited a pH optimum of 8.5 and an apparent Km for L-pipecolic acid of 1.67 x 10-3 M. L-Proline acted as a competitive inhibitor for the enzyme. The enzyme was inhibited by the sulfhydryl agents p-chloromercuribenzoate and mercuric chloride. The in vitro enzyme activity required oxygen, and upon oxidation of pipecolic acid, oxygen was reduced to hydrogen peroxide.