The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose
The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose
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DOI：
10.1073/pnas.94.5.2085
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发表时间：
1997-03-04
影响因子：
11.1
通讯作者：
Reiter, WD
中科院分区：
文献类型：
--
作者：
Bonin, CP;Potter, I;Reiter, WD
L岩藻糖是生物体中许多结构多糖和糖蛋白的活性核苷酸形式，由L岩藻糖催化合成L岩藻糖经历了4，6-脱水、3，5-差向异构化和4-还原三步反应。拟南芥突变株Mur1缺乏L-岩藻糖，在外源L-岩藻糖存在下被拯救。Gdp-L-岩藻糖合成的从头合成途径的生化分析表明，Mou1在第一个核苷酸糖相互转化步骤中被阻断，这是一个Gdp-D-甘露糖-4，6-脱水酶。从文库中克隆了一个与细菌Gdp-D-甘露糖-4，6-脱水酶序列高度连锁的表达序列标签，并在大肠杆菌中进行了表达，重组蛋白在体外具有Gdp-D-甘露糖-3，6-脱水酶活性，并能在体外补充MUR1提取液完成Gdp-L-岩藻糖的合成途径，所研究的7个MUR1等位基因均在4，6-脱水酶编码区出现单点突变，证实它代表MUR1基因。
GDP-L-fucose is the activated nucleotide sugar form of L-fucose, which is a constituent of many structural polysaccharides and glycoproteins in various organisms, The de novo synthesis of GDP-L-fucose from GDP-D-mannose encompasses three catalytic steps, a 4,6-dehydration, a 3,5-epimerization, and a 4-reduction. The mur1 mutant of Arabidopsis is deficient in L-fucose in the shoot and is rescued by growth in the presence of exogenously supplied L-fucose, Biochemical assays of the de novo pathway for the synthesis of GDP-L-fucose indicated that mur1 was blocked in the first nucleotide sugar interconversion step, a GDP-D-mannose-4,6-dehydratase. An expressed sequence tag was identified that showed significant sequence similarity to proposed bacterial GDP-D-mannose-4,6-dehydratases and was tightly linked to the mur1 locus, A full-length clone was isolated from a cDNA library, and its coding region was expressed in Escherichia coli, The recombinant protein exhibited GDP-D-mannose-3,6-dehydratase activity in vitro and was able to complement mur1 extracts in vitro to complete the pathway for the synthesis of GDP-L-fucose, All seven mur1 alleles investigated showed single point mutations in the coding region for the 4,6-dehydratase, confirming that it represents the MUR1 gene.