Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved ␣ -L -fucosyl-(1 → 2)-␤ -D -galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.

Cell walls of the Arabidopsis mutant mur2 contain less than 2% of the wild-type amount of fucosylated xyloglucan because of a point mutation in the fucosyltransferase AtFUT1. The mur2 mutation eliminates xyloglucan fucosylation in all major plant organs, indicating that Arabidopsis thaliana fucosyltransferase 1 (AtFUT1) accounts for all of the xyloglucan fucosyltransferase activity in Because the mur1 mutation affects several cell wall polysaccharides, whereas the mur2 mutation is specific to xyloglucan, the phenotypes of mur1 plants appear to be caused by structural changes in fucosylated pectic components such as rhamnogalacturonan-II. The normal growth habit and wall strength of mur2 plants casts doubt on hypotheses regarding roles of xyloglucan fucosylation in facilitating xyloglucan-cellulose interactions or in modulating growth regulator activity.

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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l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-␤-glucuronidase fusions and immunolocalization of a Fuc-containing epitope. GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed. Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant. These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types. We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme.l-Fuc is a monosaccharide constituent of various glycoproteins and polysaccharides synthesized by plant cells. It is found predominantly in xyloglucan, a hemicellulosic polysaccharide that is believed to cross-link cellulose microfibrils (Bacic et al., 1988; Carpita and Gibeaut, 1993). l-Fuc is also present in the pectic polysaccharides rhamnogalacturonan I and II and in root mucilage, which is believed to lubricate the root as it travels through the soil matrix in addition to providing protection during periods of drought (Greenland, 1979; Rougier, 1981; Baldo et al., 1983).The localization of l-fucosylated xyloglucan polymers within root cell walls has been accomplished with the use of an antibody directed against the terminal l-Fuc epitope of this hemicellulose (Puhlmann et al., 1994). These studies have shown that l-Fuc is found in almost all cells walls of the developing Arabidopsis root tip, although in different amounts (Freshour et al., 1996). More intense labeling was found in the epidermal and lateral root cap cells, which may be due to the presence of a thicker cell wall. Immunogold labeling and electron microscopy established that outer lateral root cap cell walls were heavily labeled, whereas interior-facing walls of these cells were not (Freshour et al., 1996). Terminal l-Fuc-containing epitopes were also found in most cells from mature portions of the root but were absent from the radial cross walls of endodermal cells (Freshour et al., 1996). These studies suggest that the synthesis of fucosylated polysaccharides is differentially regulated at the cellular and whole-root level in Arabidopsis. If this is the case, the de novo synthesis of l-Fuc may be tightly regulated to provide necessary precursors when and where they are needed during the development of the Arabidopsis root.The biosynthesis of l-Fuc occurs through the conversion of GDP-d-Man to GDP-l-Fuc in three catalytic steps: 4,6-dehydration...

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H 2 production pathway according to the stoichiometry CO ؉ H 2 O 3 CO 2 ؉ H 2 . Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H 2 activity was determined in the presence of the electron transport uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), the rate of H 2 production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H 2 production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H 2 reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H 2 production from gas streams containing CO.Hydrogen is a clean fuel that addresses the issues of energy security and energy independence while preserving a pristine environment. Biomass gasification generates a gas stream enriched in CO and H 2 (synthesis gas). Many microbes have been reported to metabolize CO according to the equation CO ϩ H 2 O 7 H 2 ϩ CO 2 (4, 7, 9, 25). The biological CO-to-H 2 pathway is therefore ideal if it is used following biomass gasification to convert the CO component in the synthesis gas into additional H 2 . One such candidate is the purple nonsulfur photosynthetic bacterium Rubrivivax gelatinosus CBS, which was isolated from its natural environment with the ability to metabolize CO, yielding H 2 (16). The CO oxidation pathway in R. gelatinosus CBS consists of at least two enzymatic steps: CO dehydrogenase (CODH) catalyzes the oxidation of CO, and hydrogenase mediates the reduction of protons, yielding H 2 (17), similar to their counterparts in Rhodospirillum rubrum (8,11). Earlier findings documented that both R. gelatinosus strain 1 and R. rubrum can grow in darkness by using CO as their carbon substrate (14, 24). However, the growth media used in the above studies were often supplemented with complex carboncontaining nutrients such as Trypticase, yeast extract, and sodium acetate, which complicates the conclusion that CO could serve as the sole carbon and energy source. Nonetheless, new ATP synthesis was indeed detected in R. gelatinosus strain 1 in darkness when CO was added as the carbon substrate along with Trypticase (6). This finding provided the first direct evidence in a photosynthetic bacterium that the CO-to-H 2 pathway is linked to ATP product...

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