Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Oba K, Hirai M
Department of Vegetable Breeding, National Research Institute of Vegetables, Ornamental Plants and Tea, Mie, Japan.
Plant Cell Physiol. 1998 Dec;39(12):1350-8. doi: 10.1093/oxfordjournals.pcp.a029341.
L-Galactono-gamma-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 GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDH-ase 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-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-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-gamma-lactone oxidase and L-galactono-gamma-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.
L-半乳糖酸-γ-内酯脱氢酶（EC 1.3.2.3，GLDHase）从甘薯块根线粒体中部分纯化，比活性提高了600多倍，随后通过制备型SDS-PAGE纯化了56 kDa的酶蛋白。羟基磷灰石柱纯化的GLDHase的吸收光谱在448和373 nm处有峰值，表明存在黄素作为辅基。GLDHase的活性受到石蒜碱的抑制，石蒜碱是一种在体内抑制抗坏血酸生物合成的生物碱。测定了用V8蛋白酶部分消化GLDHase产生的四个内部多肽的N端部分序列。推导的核苷酸序列用于扩增GLDHase基因的cDNA片段。该克隆编码一个由581个氨基酸残基组成的多肽，分子量为66 kDa。推导的氨基酸序列与花椰菜GLDHase的序列有77%的同一性，与大鼠的L-古洛糖酸-γ-内酯氧化酶（22%同一性）和酵母的L-半乳糖酸-γ-内酯氧化酶（17%同一性）有显著同源性，这些酶参与这些生物体中L-抗坏血酸的生物合成。吸收光谱和cDNA序列表明黄素基团是非共价结合的。我们得出结论，尽管底物和电子受体不同，但GLDHase、L-古洛糖酸-γ-内酯氧化酶和L-半乳糖酸-γ-内酯氧化酶是同源的。基因组Southern分析表明，GLDHase基因在甘薯基因组中以单拷贝形式存在。
