L-galactono-γ-lactone dehydrogenase from sweet potato:: Purification and cDNA sequence analysis
L-galactono-γ-lactone dehydrogenase from sweet potato:: Purification and cDNA sequence analysis
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DOI：
10.1093/oxfordjournals.pcp.a029341
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发表时间：
1998-12-01
影响因子：
4.9
通讯作者：
Hirai, M
中科院分区：
文献类型：
--
作者：
Imai, T;Karita, S;Hirai, M
L-半乳糖-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，比活性超过600倍，然后通过制备型SDS-PAGE纯化56 kDa的酶蛋白，羟基磷灰石柱纯化的GLDHase的吸收光谱在448处显示峰 和 373 nm，表明存在黄素作为辅基。 GLDHase 的活性受到石蒜碱的抑制，石蒜碱是一种抑制体内抗坏血酸生物合成的生物碱。确定了通过用V8蛋白酶部分消化GLDHase产生的四种内部多肽的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581个氨基酸残基的多肽，分子量为66 kDa，推导的氨基酸序列与花椰菜GLDHase的氨基酸序列有77%的同一性，与大鼠的L-古洛糖酸-γ-内酯氧化酶（22%的同一性）和酵母的L-半乳糖-γ-内酯氧化酶（17%的同一性）具有显着的同源性。 参与这些生物体中 L-抗坏血酸生物合成的酶。吸收光谱和cDNA序列表明黄素基团以非共价键结合，我们得出结论，尽管底物和电子受体不同，GLDHase、L-古洛糖酸-γ-内酯氧化酶和L-半乳糖-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。
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 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-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 accepters, Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.