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Nucleotide Sugar Interconversion
Enzymes Research:
Projects:
Background Information:
The Biosynthesis of D-Apiose in Plants:
D-Apiose is a plant-specific branched-chain monosaccharide found in rhamnogalacturonan II (RG-II), apiogalacturonan and several apioglycosides. Within RG-II, D-apiose serves as the binding site for borate, which leads to the formation of cross-links within the wall.
Biochemical studies in duckweed and parsley have established that uridine 5'-diphospho-D-apiose (UDP-D-apiose) is formed from UDP-D-glucuronate by decarboxylation and rearrangement of the carbon skeleton leading to ring contraction and branch formation.
The enzyme catalyzing this reaction also forms UDP-D-xylose by decarboxylation of UDP-D-glucuronate, and has therefore been named UDP-D-apiose/UDP-D-xylose synthase. Using a bioinformatics approach, we identified a candidate gene (AXS1) for this enzyme in Arabidopsis, and functionally expressed its cDNA in E. coli. The recombinant enzyme catalyzed the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose with a turnover number of 0.3 min-1. AXS1 required NAD+ for enzymatic activity, and was strongly inhibited by UDP-D-galacturonate. It was highly expressed in all plant organs consistent with a function in synthesizing an essential cell wall precursor. Database searches indicated the presence of closely related sequences in a variety of crop plants. The cloning of the AXS1 gene will help to investigate the biosynthesis of RG-II, and permit insights into the mechanism by which D-apiose and other branched monosaccharides are formed.
Authors:
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Michael Mølhøj, Rajeev Verma and Wolf-Dieter Reiter |
Reprint: |
| Title: |
"The biosynthesis of the branched-chain sugar D-apiose in plants: functional cloning and characterization of a UDP-D-apiose/UDP-D-xylose synthase from Arabidopsis" |
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| Journal: |
Plant J. 2003 Sep;35(6):693-703 |
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Cell Type-Specific Expression of GMDs in Arabidopsis:
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-β-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 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.
Authors:
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Bonin CP, Freshour G, Hahn MG, Vanzin GF, Reiter WD. |
Reprint: |
| Title: |
"The GMD1 and GMD2 Genes of Arabidopsis Encode Isoforms of GDP-D-Mannose 4,6-Dehydratase with Cell Type-Specific Expression Patterns." |
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| Journal: |
Plant Physiol. 2003 Jun;132(2):883-92 |
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The Biosynthesis of L-Arabinose in Plants:
The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide L-Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDP-D-Xyl to UDP-L-Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDP-D-Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDP-D-Xyl to UDP-L-Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans. Expression of MUR4-green fluorescent protein constructs in Arabidopsis revealed localization patterns consistent with targeting to the Golgi, suggesting that the MUR4 protein colocalizes with glycosyltransferases in the biosynthesis of arabinosylated cell wall components. The Arabidopsis genome encodes three putative proteins with >76% sequence identity to MUR4, which may explain why mur4 plants are not entirely deficient in the de novo synthesis of UDP-L-Ara.
Authors:
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Burget EG, Verma R, Molhoj M, Reiter WD. |
Reprint: |
| Title: |
"The Biosynthesis of L-Arabinose in Plants: Molecular Cloning and Characterization of a Golgi-Localized UDP-D-Xylose 4-Epimerase Encoded by the MUR4 Gene of Arabidopsis." |
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| Journal: |
Plant Cell 2003 Feb;15(2):523-31 |
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GER1 - A bifunctional epimerase-reductase:
L-Fucose is a monosaccharide found as a component of glycoproteins and cell wall polysaccharides in higher plants. The MUR1 gene of Arabidopsis thaliana encodes a GDP-D-mannose 4,6-dehydratase catalyzing the first step in the de novo synthesis of GDP-L-fucose from GDP-D-mannose (Bonin et al. 1997, Proc. Natl Acad. Sci. USA, 94, 2085-2090). Plant genes encoding the subsequent steps in L-fucose synthesis (3,5-epimerization and 4-reduction) have not been described previously. Based on sequence similarities to a bacterial gene involved in capsule synthesis we have cloned a gene from Arabidopsis, now designated GER1, which encodes a bifunctional 3,5-epimerase-4-reductase in L-fucose synthesis. The combined action of the MUR1 and GER1 gene products converts
GDP-D-mannose to GDP-L-fucose in vitro demonstrating that this entire nucleotide-sugar interconversion pathway could be reconstituted using plant genes expressed in Escherichia coli. In vitro assays indicated that the GER1 protein does not act as a GDP-D-mannose 3,5-epimerase, an enzymatic activity involved in the de novo synthesis of GDP-L-galactose and L-ascorbic acid. Similarly, L-ascorbate levels in GER1 antisense plants were unchanged indicating that GDP-D-mannose 3,5-epimerase is encoded by a separate gene.
Authors:
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Bonin CP, Reiter WD. |
Reprint: |
| Title: |
"A bifunctional epimerase-reductase acts downstream of the MUR1 gene product and completes the de novo synthesis of GDP-L-fucose in Arabidopsis." |
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| Journal: |
Plant J 2000 Mar;21(5):445-54 |
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MUR1 gene catalyzes the first step in the de novo synthesis of GDP-L-fucose:
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.
Authors:
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Bonin CP, Potter I, Vanzin GF, Reiter WD. |
Reprint: |
| Title: |
"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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| Journal: |
Proc Natl Acad Sci USA 1997 Mar 4;94(5):2085-90 |
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Nucleotide Sugar Interconversion Enzymes
Background:
Nucleotide sugar interconversion pathways represent
a series of enzymatic reactions by which plants synthesize activated
monosaccharides for the incorporation into cell wall polysaccharides,
glycolipids, glycoproteins and low-molecular weight glycoconjugates.
Although biochemical aspects of these metabolic pathways are reasonably
well understood, the identification and characterization of genes
encoding nucleotide sugar interconversion enzymes is still in
its infancy. Arabidopsis mutants defective in the activation
and interconversion of specific monosaccharides have recently
become available, and several genes in these pathways have been
cloned and characterized. An evaluation of the Arabidopsis
databases suggests that the majority of these enzymes are encoded
by small gene families, and that most of these coding regions
are transcribed. Although most of the putative proteins are predicted
to be soluble, others contain N-terminal extensions encompassing
a transmembrane domain. This suggests that some nucleotide sugar
interconversion enzymes are targeted to an endomembrane system,
such as the Golgi apparatus, where they may co-localize with glycosyltransferases
in cell wall synthesis. The functions of the predicted coding
regions can most likely be established via reverse genetic approaches
and the expression of proteins in heterologous systems. The genetic
characterization of nucleotide sugar interconversion enzymes has
the potential to understand the regulation of these complex metabolic
pathways and to permit the modification of cell wall material
by changing the availability of monosaccharide precursors.
(For more detailed information regarding sugar-nucleotide interconversion pathways, view paper below.)
Authors:
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Reiter WD, Vanzin GF. |
Reprint: |
| Title: |
"Molecular genetics of nucleotide sugar interconversion pathways in plants." |
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| Journal: |
Plant Mol Biol 2001 Sep;47(1-2):95-113 |
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