Conklin Patricia L, Gatzek Stephan, Wheeler Glen L, Dowdle John, Raymond Marjorie J, Rolinski Susanne, Isupov Mikhail, Littlechild Jennifer A, Smirnoff Nicholas
Department of Biological Sciences, State University of New York, Cortland, New York 13045, USA.
J Biol Chem. 2006 Jun 9;281(23):15662-70. doi: 10.1074/jbc.M601409200. Epub 2006 Apr 4.
In plants, a proposed ascorbate (vitamin C) biosynthesis pathway occurs via GDP-D-mannose (GDP-D-Man), GDP-L-galactose (GDP-L-Gal), and L-galactose. However, the steps involved in the synthesis of L-Gal from GDP-L-Gal in planta are not fully characterized. Here we present evidence for an in vivo role for L-Gal-1-P phosphatase in plant ascorbate biosynthesis. We have characterized a low ascorbate mutant (vtc4-1) of Arabidopsis thaliana, which exhibits decreased ascorbate biosynthesis. Genetic mapping and sequencing of the VTC4 locus identified a mutation (P92L) in a gene with predicted L-Gal-1-P phosphatase activity (At3g02870). Pro-92 is within a beta-bulge that is conserved in related myo-inositol monophosphatases. The mutation is predicted to disrupt the positioning of catalytic amino acid residues within the active site. Accordingly, L-Gal-1-P phosphatase activity in vtc4-1 was approximately 50% of wild-type plants. In addition, vtc4-1 plants incorporate significantly more radiolabel from [2-(3)H]Man into L-galactosyl residues suggesting that the mutation increases the availability of GDP-L-Gal for polysaccharide synthesis. Finally, a homozygous T-DNA insertion line, which lacks a functional At3g02870 gene product, is also ascorbate-deficient (50% of wild type) and deficient in L-Gal-1-P phosphatase activity. Genetic complementation tests revealed that the insertion mutant and VTC4-1 are alleles of the same genetic locus. The significantly lower ascorbate and perturbed L-Gal metabolism in vtc4-1 and the T-DNA insertion mutant indicate that L-Gal-1-P phosphatase plays a role in plant ascorbate biosynthesis. The presence of ascorbate in the T-DNA insertion mutant suggests there is a bypass to this enzyme or that other pathways also contribute to ascorbate biosynthesis.
在植物中，一种推测的抗坏血酸（维生素C）生物合成途径是通过GDP-D-甘露糖（GDP-D-Man）、GDP-L-半乳糖（GDP-L-Gal）和L-半乳糖进行的。然而，植物中从GDP-L-Gal合成L-半乳糖所涉及的步骤尚未完全明确。在此，我们提供了L-半乳糖-1-磷酸磷酸酶在植物抗坏血酸生物合成中体内作用的证据。我们对拟南芥的一个低抗坏血酸突变体（vtc4-1）进行了表征，该突变体表现出抗坏血酸生物合成减少。对VTC4基因座的遗传定位和测序确定了一个具有预测的L-半乳糖-1-磷酸磷酸酶活性的基因（At3g02870）中的一个突变（P92L）。第92位脯氨酸位于相关肌醇单磷酸酶中保守的β-凸起内。该突变预计会破坏活性位点内催化氨基酸残基的定位。因此，vtc4-1中的L-半乳糖-1-磷酸磷酸酶活性约为野生型植物的50%。此外，vtc4-1植物从[2-(3)H]甘露糖中掺入到L-半乳糖基残基中的放射性标记明显更多，这表明该突变增加了GDP-L-Gal用于多糖合成的可用性。最后，一个缺乏功能性At3g02870基因产物的纯合T-DNA插入系也缺乏抗坏血酸（为野生型的50%）且缺乏L-半乳糖-1-磷酸磷酸酶活性。遗传互补试验表明插入突变体和VTC4-1是同一基因座的等位基因。vtc4-1和T-DNA插入突变体中抗坏血酸显著降低以及L-半乳糖代谢紊乱表明L-半乳糖-1-磷酸磷酸酶在植物抗坏血酸生物合成中起作用。T-DNA插入突变体中存在抗坏血酸表明存在该酶的旁路途径，或者其他途径也对抗坏血酸生物合成有贡献。
