Role of Pipecolic Acid in the Biosynthesis of Lysine in Rhodotorula glutinis

Abstract

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.

Use of α‐aminoadipate and lysine as sole nitrogen source by Schizosacharomyces pombe and selected pathogenic fungi

“…Such mutants exhibit lysine auxotrophy, and belong largely to the lys2 locus (CHATTOO et al 1979, BHATTACHARJEE 1985. Although lysine mutants blocked before the AA step (lys4, lys6, lys7, lys8, lysl0, lys15, and lys16) of S. cerevisiae grow on AA supplemented medium ( 5 mg ammonium sulfate and 10 to 50 pg AA per ml medium), only lys2, lys5, and lys14 mutants, blocked after the AA step can grow on medium containing AA as the sole nitrogen source (2 to 5 mg AA and 10 to 50 pg lysine per ml medium; CHATOO et al 1979, BORELL et al 1984. The ability of the fission yeast Schizosaccharomyces pombe and pathogenic fungi to use AA as sole nitrogen source has never been investigated.…”

Lysine degradation in Pichia guilliermondii: Characterization of a novel enzyme, L‐lysine:pyruvate aminotransferase

“…Enzyme assay: L-Lysine: pyruvate aminotransferase (Lys-AT) was assayed by determining the amount of a-aminoadipate-&semialdehyde formed (KINZEL and BHATTACHARJEE 1979). The reaction mixture (standard condition) contained 20 mM L-lysine, 5 mM pyruvate, 0.5 mM pyridoxal-5-phosphate, 100 mM TrisiHCl buffer (pH 9.0), and enzyme solution (10 pg of the DEAE-cellulose eluate) in a final volume of 1 ml.…”

Role of L-lysine-alpha-ketoglutarate aminotransferase in catabolism of lysine as a nitrogen source for Rhodotorula glutinis

“…Cultures were grown at 28°C with vigorous shaking, and the culture supernatants of cells grown in different nitrogen source media were examined for the accumulation of ASA as a p-dimethylaminobenzaldehyde-reactive product (9, 16). The accumulation product was further characterized on the basis of the absorption spectra (9).…”

“…Dialyzed extracts were prepared, and pipecolic acid oxidase and saccharopine dehydrogenase (EC 1.5.1.7) activities as well as protein were determined by procedures described previously (9,11). Lysine-a-ketoglutarate aminotransferase (LKGT) was assayed by determining the amount of ASA formed (9,19).…”

“…Dialyzed extracts were prepared, and pipecolic acid oxidase and saccharopine dehydrogenase (EC 1.5.1.7) activities as well as protein were determined by procedures described previously (9,11). Lysine-a-ketoglutarate aminotransferase (LKGT) was assayed by determining the amount of ASA formed (9,19). The reaction mixture consisted of the following: pyridoxal phosphate, 0.125 mM; L-lysine, 2.5 mM; a-ketoglutarate, 2.5 mM; 0.25 M potassium phosphate buffer (pH 7.5); and dialyzed extract equivalent to 0.5 to 1 mg of protein in a final volume of 1.0 ml.…”