D-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae

D-Arabinono-1,4-lactone oxidase catalysing the final step of D-erythroascor bic acid biosynthesis was purified from the mitochondrial fraction of Sacch aromyces cerevisiae. Based on the amino acid sequence analysis of the enzym e, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme. The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59493Da, The deduce d amino acid sequence of the enzyme shared 32% and 21% identity with that o f L-gulono-1,4-lactone oxidase from rat and L-galactono-1,4-lactone dehydro genase from cauliflower, respectively, and contained a putative transmembra ne segment and a covalent FAD binding site. Blot hybridization analyses sho wed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8 kb in size. In the alo1 mutants, D-erythroas corbic acid and the activity of D-arabinono-1,4-lactone oxidase could not b e detected. The intracellular concentration of D-erythroascorbic acid and t he enzyme activity increased up to 6.9-fold and 7.3-fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid. The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress.