Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.
Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.
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DOI：
10.1074/jbc.m109.069369
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发表时间：
2010-01-29
期刊：
影响因子：
--
通讯作者：
Tenhaken R
中科院分区：
文献类型：
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作者：
Pieslinger AM;Hoepflinger MC;Tenhaken R
核苷酸糖是植物细胞壁中碳水化合物聚合物的结构单元。它们由糖-1-磷酸合成或差向异构化为核苷酸糖。初生细胞壁的主要前体是UDP-葡萄糖醛酸，它可以通过两种独立的途径合成。一个起始于肌醇环裂解成葡萄糖醛酸，其需要葡萄糖醛酸激酶和焦磷酸化酶活化成UDP-葡萄糖醛酸。在这里，我们报道了从百合花粉中纯化葡萄糖醛酸激酶的方法。结合六个色谱步骤纯化40-kDa蛋白质，并对肽进行从头测序。这允许从拟南芥中克隆该基因并在大肠杆菌中表达重组蛋白用于生化表征。葡萄糖醛酸激酶（Glucuronokinase，GHMP）是GHMP激酶超家族的新成员，对D-葡萄糖醛酸具有独特的底物特异性，Km为0.7mm，需要ATP作为磷酸供体（Km为0.56mm）。在拟南芥中，该基因在所有植物组织中表达，偏好花粉。葡萄糖醛酸激酶的基因存在于（所有）植物、一些藻类和一些细菌以及一些低等动物中。
Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a Km of 0.7 mm. It requires ATP as phosphate donor (Km 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.