Journal Article

L-Galactono-γ-lactone Dehydrogenase from Sweet Potato: Purification and cDNA Sequence Analysis

Tsuyoshi Imai, Shuichi Karita, Gen-ichi Shiratori, Miyo Hattori, Tsukasa Nunome, Kazuko Ôba and Masashi Hirai

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 39, issue 12, pages 1350-1358
Published in print December 1998 | ISSN: 0032-0781
Published online December 1998 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029341
L-Galactono-γ-lactone Dehydrogenase from Sweet Potato: Purification and cDNA Sequence Analysis

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L-Galactono-γ-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE. The absorption spectrum of the hydroxylapatite column-purified GLDHase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDHase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo. N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined. The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene. The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa. The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-γ-lactone oxidase (22% identity) from rat and L-galactono-γ-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms. The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently. We conclude that GLDHase, L-gulono-y-lactone oxidase and L-galactono-γ-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors. Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.

Keywords: L-Ascorbic acid; cDNA sequence; L-Galact-ono-γ-lactone dehydrogenase (EC 1.3.2.3); Sweet potato (Ipomoea batatas)

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Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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