D-arabinose dehydrogenase and biosynthesis of erythroascorbic acid in Candida albicans.
D-Arabinose dehydrogenase was purified 2750-fold from the cytosolic fraction of Candida albicans to apparent homogeneity, with an overall yield of 3%, by a purification procedure consisting of ammonium sulfate precipitation and DEAE-Sepharose A-50, Sephacryl S-200, Cibacron blue and phenyl-Sepharose CL-4B chromatographies. Gel-filtration chromatography gave an apparent molecular mass of 41 kDa and SDS-PAGE showed only one protein band corresponding to a molecular mass of 42 kDa, indicating that the enzyme is a single polypeptide. The enzyme was optimally active at pH 8.0 and the pI value of the enzyme was 5.0. The enzyme was relatively stable from pH 4.5 to 7.5. The optimal temperature for the enzyme activity was 30 degrees C. The activity of the enzyme was inhibited by Hg2+, Fe2+, Zn2+, Cu2+, Mg2+, Mn2+, N-ethylmaleimide and p-chloromercuribenzoic acid. The enzyme catalysed the oxidation of D-arabinose, L-fucose, L-xylose and L-galactose, which have the same configurations of hydroxyl groups at C2- and C3-positions, with apparent K(m) values of 29.2, 28.9, 37.1 and 91.3 mM at pH 8.0, respectively, with 50 microM NADP+. The enzyme used NADP+ as a coenzyme. Apparent K(m) value at 60 mM D-arabinose for NADP+ was 44.6 microM. NADPH inhibited the enzyme activity competitively with respect to NADP+ (Ki = 78.6 microM). The amino-terminal sequence of the enzyme was Met-Lys-Leu-Ala-Thr-Glu-Ile-Asp-Phe-X-Leu-Asn-Asn-Gly-. The reaction product was D-arabinono-1,4-lactone, judged from gas-liquid chromatography/mass spectrometry. In C. albicans, D-erythroascorbic acid was formed from D-arabinose by D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase.
Kim
S T
ST
Department of Microbiology, College of Natural Sciences, Seoul National University, South Korea.
Huh
W K
WK
Kim
J Y
JY
Hwang
S W
SW
Kang
S O
SO
D000595:Amino Acid Sequence; D001089:Arabinose / Q000378:metabolism; D001205:Ascorbic Acid / Q000096:biosynthesis*; D002176:Candida albicans / Q000201:enzymology* / Q000378:metabolism; D002413:Cations, Divalent; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009005:Monosaccharides / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D020245:p-Chloromercuribenzoic Acid
D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D004791:Enzyme Inhibitors; D009005:Monosaccharides; C005056:isoascorbic acid; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D001089:Arabinose; D013401:Sugar Alcohol Dehydrogenases; C028590:D-arabinose dehydrogenase; C090492:D-arabinonolactone oxidase; D005033:Ethylmaleimide; D001205:Ascorbic Acid