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-Dmannose-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.L-Fucose (6-deoxy-L-galactose) is a monosaccharide found in a diverse array of organisms. The sugar is a known component of bacterial lipopolysaccharides, mammalian and plant glycoproteins, and polysaccharides of plant cell walls such as xyloglucan and rhamnogalacturonans I and II. The precise function of L-fucose within these polysaccharides is not clear, but it may stabilize conformations of xyloglucan, which can efficiently bind to cellulose microfibrils (1), possibly aiding in cell wall integrity. Furthermore, xyloglucan fucosylation is essential for the biological activity of some xyloglucan-derived oligosaccharides (2). The pathway for the synthesis of L-fucose has been studied biochemically, but genes for the corresponding enzymes have not been cloned from any eukaryote.GDP-L-fucose (guanosine-diphospho-L-fucose) is the activated form of this sugar, synthesized de novo from GDP-Dmannose via a three-step mechanism or through a salvage pathway involving phosphorylation of free L-fucose and subsequent nucleoside-diphosphate attachment (3-5). The de novo pathway for GDP-L-fucose production is shown in Fig. 1. The first step is catalyzed by GDP-D-mannose-4,6-dehydratase and involves the formation of the intermediate GDP-4-keto-6-deoxy-D-mannose, which is then used in the second and third steps of the pathway by 3,5-epimerase and 4-reductase activities to yield GDP-L-fucose. The pathway was initially elucidated in bacteria but has since been characterized in mammalian and plant systems (6)(7)(8)(9)(10)(11).Recently an L-fucose-deficient cell wall mutant, mur1, was isolated from Arabidopsis thaliana and characterized phenotypically (12). Eight recessive mur1 alleles were obtained from this screen, most of which exhibit 50-to 200-fold reducti...

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