The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.
The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.
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DOI：
10.1093/plphys/kiab010
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发表时间：
2021-04-23
期刊：
影响因子：
7.4
通讯作者：
Botella MA
中科院分区：
文献类型：
--
作者：
Fenech M;Amorim-Silva V;Esteban Del Valle A;Arnaud D;Ruiz-Lopez N;Castillo AG;Smirnoff N;Botella MA
参与光合生物中l-抗坏血酸生物合成的酶（Smirnoff-Wheeler [SW]途径）已得到充分确立。在这里，我们分析了它们的亚细胞定位和潜在的物理相互作用，并评估了它们在控制抗坏血酸合成中的作用。本氏烟草和拟南芥突变体中SW基因的C末端标记融合体与基因组构建体互补的瞬时表达表明，虽然GDP-d-甘露糖差向异构酶是胞质的，但从GDP-d-甘露糖焦磷酸化酶（GMP）到l-半乳糖脱氢酶（l-GalDH）的所有酶都显示出胞质/核双重定位。所有表达由其内源启动子驱动的功能性SW蛋白绿色荧光蛋白融合体的转基因株系显示融合蛋白的高积累，除了表达具有非常低丰度的GDP-1-半乳糖磷酸化酶（GGP）蛋白的那些株系。SW途径酶的单个或组合在N.如果包括GGP，则本塞姆那仅增加抗坏血酸浓度。虽然我们没有检测到直接的相互作用的途径，使用酵母双杂交分析，连续SW酶，以及第一个和最后一个酶（GMP和L-GalDH）在免疫共沉淀研究。凝胶过滤色谱法支持这种关联，显示SW蛋白在高分子量组分中的存在。最后，代谢控制分析，结合已知的动力学特征表明，先前报道的反馈阻遏在GGP步骤，结合其相对较低的丰度，赋予高通量控制系数和合理化为什么其他酶的操作对抗坏血酸浓度的影响不大。代谢工程、遗传分析和功能突变互补鉴定了GDP-1-半乳糖磷酸化酶作为绿色组织中抗坏血酸生物合成的主要控制点。
The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff–Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration. Metabolic engineering, genetic analysis, and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.