NADP(+)-dependent D-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae.
The molecular aspects and physiological significance of NADP(+)-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP(+)-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H(2)O(2) and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H(2)O(2) with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.
Amako
Katsumi
K
Faculty of Nutrition, Kobe Gakuin University. amakok@nutr.kobegakuin.ac.jp
Fujita
Kazuyo
K
Iwamoto
Chiaki
C
Sengee
Myagmarsuren
M
Fuchigami
Kazuko
K
Fukumoto
Junko
J
Ogishi
Yasuka
Y
Kishimoto
Ritsuko
R
Goda
Kiyoshi
K
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000096:biosynthesis*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; C028590:D-arabinose dehydrogenase; D001205:Ascorbic Acid