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

Abstract

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.