Characterisation of D-arabinono-1,4-lactone oxidase from Candida albicans ATCC 10231.
D-Erythroascorbic acid was detected from the cell extracts of a dimorphic fungus, Candida albicans. Its concentration in yeast cells grown at 25 degrees C was estimated to be about 0.45 mumol/ml cell water. D-Arabinono-1,4-lactone oxidase, which catalyses the final step in the biosynthesis of D-erythroascorbic acid, was purified 639-fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Triton X-100 solubilisation, ammonium sulphate precipitation, anion-exchange, hydrophobic-interaction, gel-filtration and dye-ligand chromatographies. Gel-filtration chromatography and polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave apparent molecular masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only one protein band corresponding to a molecular mass of 66.7 kDa. Considering the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spectrum of a flavin-containing enzyme. The flavin was not released by treatment with SDS, CCl3CO2H or boiling, indicating that it may be covalently bound to the enzyme protein. The enzyme was optimally active at 40 degrees C and at pH 6.1. The enzyme was stable in the range pH 7.5-10. An apparent Km value for D-arabinono-1,4-lactone was 44.1 mM. L-Galactono-1,4-lactone, L-gulono-1,4-lactone and L-xylono-1,4-lactone could also serve as substrates. Competitive inhibition was demonstrated with D-glucono-1,5-lactone, L-arabinono-1,4-lactone, D-galactono-1,4-lactone and D-gulono-1,4-lactone. p-Chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd2+, Hg2+, Mn2+ and Zn2+ exhibited inhibitory effects on the enzyme.
Huh
W K
WK
Department of Microbiology, College of Natural Sciences, Seoul National University, Republic of Korea.
Kim
S T
ST
Yang
K S
KS
Seok
Y J
YJ
Hah
Y C
YC
Kang
S O
SO
D001205:Ascorbic Acid / Q000096:biosynthesis; D001667:Binding, Competitive; D002176:Candida albicans / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
C005056:isoascorbic acid; D013401:Sugar Alcohol Dehydrogenases; C090492:D-arabinonolactone oxidase; D001205:Ascorbic Acid