The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-d-mannose-46-dehydratase catalyzing the first step in the de novo synthesis of GDP-l-fucose

摘要

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-4,6-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.