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Biochemical genetics of nucleotide sugar interconversion reactions
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Biochemical genetics of nucleotide sugar interconversion reactions Wolf-Dieter Reiter During the past few years, substantial progress has been made to understand the enzymology and regulation of nucleotide sugar interconversion reactions that are irreversible in vivo on thermodynamic grounds. Feedback inhibition of enzymes by metabolic end products appears to be a common theme but some experimental results on recombinant enzymes are difficult to interpret. Using a combination of metabolic flux analysis, enzyme assays, and bioinformatics approaches, the significance of several proposed alternate pathways has been clarified. Expression of plant nucleotide sugar interconversion enzymes in yeast has become a promising approach to understand metabolic regulation and produce valuable compounds. In a major advance for the understanding of the synthesis of arabinosylated cell wall polysaccharides, reversibly glycosylated proteins turned out to act as mutases that interconvert the pyranose and furanose forms of UDP-L-arabinose. Addresses Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, USA Corresponding author: Reiter, Wolf-Dieter ([email protected])
Current Opinion in Plant Biology 2008, 11:236–243 This review comes from a themed issue on Physiology and metabolism Edited by Ken Keegstra and Markus Pauly
1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.03.009
Introduction Most of the carbon that is photosynthetically fixed by plants is incorporated into cell wall material where it remains throughout the plant’s life cycle. This makes plant cell wall polysaccharides the major source of renewable biomass that can be used for the generation of ethanol via fermentation processes [1]. Because naturally occurring cell wall material is highly recalcitrant to enzymatic degradation, there is great interest in exploring possibilities to improve its deconstruction via transgenic approaches. Because glycosyltransferases in cell wall synthesis use nucleotide sugars as monosaccharide donors [2–5], modifications in the abundance of these precursors via transgenic approaches are likely to cause changes in cell wall composition that may improve the digestibility and abundance of cell wall polymers. Most genes encodCurrent Opinion in Plant Biology 2008, 11:236–243
ing nucleotide sugar interconversion enzymes have been cloned and characterized via biochemical, genetic and bioinformatics approaches, and the reader is referred to the review by Seifert [3] for coverage of the literature up to early 2004. The current review will focus on novel developments regarding carbon fluxes through nucleotide sugar interconversion pathways and the discovery of new genes.
Flux of carbon from primary photosynthesis products to cell wall material Carbon fixation in the chloroplasts leads to the formation of fructose 6-phosphate as the first phosphorylated hexose from which virtually all building blocks for the synthesis of cell wall material are derived (Figure 1). The combined action of phosphosugar isomerases and mutases yield glucose-1-phosphate and mannose-1-phosphate that are activated via cytosolic pyrophosphorylases to yield the nucleotide sugars UDP-D-glucose (UDP-Glc) and GDPD-mannose (GDP-Man) [2]. Virtually all cell wall precursors are derived from these two nucleotide sugars via a series of mechanistically related reactions that are catalyzed by 4-epimerases, 3,5-epimerases, 4-reductases, 4,6dehydratases, dehydrogenases, and decarboxylases [2,3]. With the exception of the nucleotide sugar dehydrogenases that act on carbon 6 of the sugar moiety, all of these enzymes initiate the catalytic cycle via oxidation of carbon 4 to a keto group with the help of an NAD(P)+ cofactor that is transiently reduced to NAD(P)H, and subsequently re-oxidized to its original state. The bestknown enzyme in this group is UDP-Glc 4-epimerase, which occurs in most organisms to either synthesize or degrade D-galactose. Extensive studies on the genetics and the biochemical properties of UDP-Glc 4-epimerase isoforms from Arabidopsis [6,7] and barley [8] have recently been published, and the reader is referred to these papers for detailed information. All of the epimerization reactions are freely reversible but the conversion of UDP-Glc to UDP-D-glucuronate (UDP-GlcA) [9,10] and the subsequent decarboxylation of this nucleotide sugar to UDP-D-xylose (UDP-Xyl) [11,12] are too exergonic to be reversible under in vivo conditions. This is an important issue when considering the regulation of carbon flux, in particular because sugar moieties derived from UDP-GlcA are abundant components of cell wall polymers including both pectins and hemicelluloses. For instance, D-galacturonate (GalA) is the most abundant monosaccharide in pectic cell wall material, and D-xylose (Xyl) is the predominant monosaccharide in xylans (Table 1). Like the formation of www.sciencedirect.com
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Table 1 UDP- and GDP-sugars, and the main cell wall components into which their monosaccharide moieties are incorporated Nucleotide sugar
Polysaccharide(s)
UDP-a-D-glucose UDP-a-D-galactose UDP-a-D-glucuronate UDP-a-D-galacturonate UDP-a-D-xylose UDP-a-D-apiose UDP-b-L-arabinose UDP-b-L-rhamnose GDP-a-D-glucose GDP-a-D-mannose GDP-b-L-galactose GDP-b-L-fucose
Cellulose (b-1 ! 4), callose (b-1 ! 3), mixed-linked glucans (b-1 ! 3 and b-1 ! 4), xyloglucan (b-1 ! 4) Rhamnogalacturonan-I and arabinogalactan-proteins (b-1 ! 3, b-1 ! 4, b-1 ! 6), xyloglucan (b-1 ! 2) Glucurono(arabino)xylans (a-1 ! 2), rhamnogalacturonan-II (b-1 ! 4) Homogalacturonan (a-1 ! 4), rhamnogalacturonan-I (a-1 ! 2), rhamnogalacturonan-II (a-1 ! 2, a-1 ! 3, a-1 ! 4) Xylans (b-1 ! 4), xyloglucan (a-1 ! 6) Rhamnogalacturonan-II (b-1 ! 2) Rhamnogalacturonan-I and arabinogalactan-proteins (a-1 ! 3, a-1 ! 5), (glucurono)arabinoxylans (a-1 ! 2, a-1 ! 3) Rhamnogalacturonan-I (a-1 ! 4), rhamnogalacturonan-II (b-1 ! 3, a-1 ! 2, a-1 ! 3, a-1 ! 5) Glucomannans (b-1 ! 4) (Gluco)mannans (b-1 ! 4) Rhamnogalacturonan-II (a-1 ! 2) Rhamnogalacturonan-II (a-1 ! 2, a-1 ! 4), xyloglucan (a-1 ! 2)
The type(s) of linkages that connect a specific sugar to other parts of the respective polysaccharides are shown in parentheses.
UDP-GlcA, the synthesis of UDP-L-rhamnose (UDPRha) from UDP-Glc and the conversion of GDP-Man to GDP-L-fucose (GDP-Fuc) are irreversible reactions under in vivo conditions, and the respective biosynthetic pathways are expected to be tightly regulated. Although the pathways shown in Figure 1 are well documented, some alternate routes toward the synthesis of nucleotide sugars have been proposed. For instance, a hypothetical 2-epimerization of GDP-Man [13] would lead to the formation of GDP-Glc that is used by glucomannan synthases in vitro [14]. This pathway would be an alternative to the synthesis of GDP-Glc from glucose-1phosphate and GTP via a pyrophosphorylase. Another unresolved issue is whether at least some UDP-GalA is formed by the dehydrogenation of UDP-Gal as described by Steward and Copeland [15] instead of the well-established 4-epimerization of UDP-GlcA. Sharples and Fry [16] recently addressed these issues by feeding suspension-cultured Arabidopsis cells a mixture of tritiated galactose and 14C-labeled fructose, and following the incorporation of the radiolabels into nucleotide sugars and cell wall polymers as a function of time. Galactose and fructose enter nucleotide sugar interconversion pathways from opposite ends of a central metabolic pathway (Figure 1), which permits an evaluation of carbon fluxes through alternate pathways by determining the 3H/14C ratio in nucleotide sugars and cell wall-derived monosaccharides [16]. The experimental data indicate that GDP-Glc is primarily formed by the action of a pyrophosphorylase, and that UDP-GalA is primarily formed by 4epimerization of UDP-GlcA, a reaction that occurs within the endomembrane system [17–19]. These results do not rule out the possibility that the proposed alternate pathways operate at low capacity in the suspension-cultured cells but at a higher capacity in specific cell types of intact plants. Nonetheless, the apparent absence of UDP-Gal dehydrogenase activity is consistent with the observation that recombinant UDP-Glc dehydrogenase from soybean [9] and three of the four isoforms of UDP-Glc dehydrowww.sciencedirect.com
genase from Arabidopsis do not act on UDP-Gal (the remaining isoform UGD1 was not tested) [10]. Furthermore, the Arabidopsis genome does not contain closely related sequences that would qualify as candidate genes for UDP-Gal dehydrogenase, and no sequence entries for UDP-Gal dehydrogenase exist in the NCBI databases. All of this suggests that this enzymatic activity is absent from all major groups of organisms including plants. The controversy regarding the formation of GDP-Glc is more difficult to address via bioinformatics approaches because neither GDP-Glc pyrophosphorylase nor the hypothetical GDP-Man 2-epimerase have been cloned from plants. Some GDP-Man pyrophosphorylases such as the enzyme from a strain of Escherichia coli have been shown to synthesize GDP-Glc from glucose-1-phosphate and GTP [20], and the same may be true for the corresponding enzymes from plants. A study on partially purified GDP-Glc pyrophosphorylase from pea seedlings indicated that mannose-1-phosphate is not a substrate for this enzyme [21] but the gene encoding this enzyme remains to be cloned. It would be interesting to determine whether any of the three predicted GDP-Man pyrophosphorylases in the Arabidopsis genome accept glucose-1-phosphate as a substrate. Leaky mutations in one of the GDP-Man pyrophosphorylase isoforms cause a reduction in ascorbic acid content (vtc1) [22], and tight mutations in the same gene (cyt1) are seedling-lethal presumably because of a defect in the N-glycosylation of cellulose synthases [23]. This strongly suggests that the VTC1/CYT1 gene is the predominant GDP-Man pyrophosphorylase in Arabidopsis whereas the functions of its two homologs are unknown. The work by Sharples and Fry [16] also addresses the flux through a well-known alternate pathway toward the synthesis of UDP-GlcA, namely the oxidation of myoinositol to GlcA via myo-inositol oxygenase (MIOX) [24]. The GlcA formed in this reaction is then converted to UDP-GlcA via a salvage pathway (blue-boxed area in Current Opinion in Plant Biology 2008, 11:236–243
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Figure 1
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Figure 1). In the suspension-cultured cell system, flux through this pathway appeared to be very low compared to the oxidation of UDP-Glc to UDP-GlcA but the situation may be very different in other cell types or plant species. For example, there are large differences in the expression levels of the known [25] or predicted MIOX isoforms in Arabidopsis, and in the number of MIOX-related ESTs between plant species [26]. It is unclear at this time whether the MIOX pathway makes any significant contribution toward the synthesis of cell wall material or whether it is simply involved in the turnover of myo-inositol that is primarily used for the synthesis of phospholipids or galactinol, the precursor for the production of raffinose family oligosaccharides [27].
activities of UGD1 and UXS3 in Arabidopsis cells were similar to those in the transgenic yeast line, this would raise the question how plant cells maintain an adequate pool of UDP-GlcA for incorporation into cell wall material and the synthesis of UDP-GalA within the Golgi. It appears that the activity of the various cytosolic and membrane-bound UDP-Xyl synthase isoforms needs to be tightly regulated to circumvent problems. It could be argued that a high rate of transport of UDP-GlcA across the Golgi membrane would shield it from decarboxylation by cytosolic UDP-Xyl synthase activity but several isoforms of UDP-Xyl synthase are targeted to the endomembrane system [2,11] where they are expected to compete with UDP-GlcA 4-epimerases and glucuronosyltransferases for their common substrate UDP-GlcA.
A gene encoding UDP-Glc dehydrogenase was initially cloned from soybean [28] but more recent studies focused on the four-member UDP-Glc dehydrogenase gene family from Arabidopsis. Oka and Jigami [29] functionally expressed the UGD1 isoform of this enzyme in the yeast Saccharomyces cerevisiae either alone or in combination with the cytosolic UXS3 isoform of UDP-Xyl synthase from Arabidopsis. Because wild type yeast contains neither enzyme and does not utilize the respective end products, it is an ideal system to overproduce rare nucleotide sugars, and to study the regulation of enzymatic activities both in vivo and in vitro. Expression of UGD1 alone caused massive accumulation of UDP-GlcA and an approximately 50% reduction in the pool of UDPGlc. Complete conversion of UDP-Glc to UDP-GlcA was presumably prevented by product inhibition of the enzyme with a Ki value of 99 mM [29]. In yeast cells overexpressing both UDP-Glc dehydrogenase and UDPXyl synthase, the pools of UDP-Glc and UDP-Xyl were similar to each other, and no depletion of UDP-Glc was observed. This is presumably because of the strong competitive inhibition of UGD1 by UDP-Xyl with a Ki value of 4.9 mM [29]. Recombinant UDP-Glc dehydrogenase from soybean shows similar inhibition by UDPXyl with a Ki value of 10 mM [9] but the UGD2, UGD3 and UGD4 isoforms from Arabidopsis are only weakly inhibited by UDP-Xyl [10]. Yeast cells expressing both UDP-Glc dehydrogenase and UDP-Xyl synthase did not contain detectable amounts of UDP-GlcA indicating that this substrate is rapidly metabolized to UDP-Xyl. If the
The biosynthesis of UDP-L-rhamnose: consolidating three enzymes into one In prokaryotes, dTDP-D-glucose (dTDP-Glc) rather than UDP-Glc is set aside for the synthesis of activated Lrhamnose to separate the nucleotide sugar pools for central metabolic pathways from those for the synthesis of capsular polysaccharides [30,31]. The three steps in this bacterial interconversion reaction are catalyzed by three separate enzymes, a 4,6-dehydratase, a 3,5-epimerase, and a 4-reductase that are usually referred to as RmlB, RmlC and RmlD, respectively [32] (Figure 2). In plants, biochemical aspects of the analogous conversion of UDPGlc to UDP-Rha have been unclear because the enzyme was never purified to homogeneity although fractionated cell extracts have been used to synthesize UDP-Rha in vitro [33]. An evaluation of sequence similarities between the bacterial Rml proteins and the Arabidopsis genome sequence led to the proposal that there are three genes (RHM1, RHM2 and RHM3) encoding proteins where a 4,6-dehydratase domain is translationally fused to a combined 3,5-epimerase-4-reductase domain [2]. A separate coding region (UER1) is highly homologous to the 3,5epimerase-4-reductase domain of the RHM proteins but does not encompass the 4,6-dehydratase domain. To test the proposed function of the RHM proteins, Oka et al. [34] recombinantly expressed the RHM2 protein and its two predicted subdomains in yeast, and measured nucleotide sugar interconversion activities. The RHM2 isoform was chosen because mutants in this gene had been shown to be defective in the production of seed coat mucilage
(Figure 1 Legend ) Overview of the main nucleotide sugar interconversion reactions in plants, and their relationship to central pathways in carbohydrate metabolism. Sugar phosphates in central metabolic pathways are boxed in orange. Mannose-1-phosphate (upper part of orange box) is activated to GDP-D-Man, which is the precursor of GDP-sugars except for GDP-D-Glc. Glucose-1-phosphate (lower part of the orange box) is activated to UDP-D-Glc, which is converted into other UDP-sugars via branched pathways. Single arrows denote reactions that are essentially irreversible in vivo, and double arrows denote reactions that are expected to be close to equilibrium. Some of the salvage pathways for free monosaccharides are included in this figure. Note that the salvage pathway for D-GlcA connects the oxidative breakdown of myo-inositol to the formation of nucleotide sugars (blue box). Bars across arrows indicate known mutations in Arabidopsis that affect at least one isoform of an interconversion enzyme. hsr8 (high sugar response8; lower right corner) is a recently isolated mutant allele of the MUR4-encoded UDP-D-Xyl 4-epimerase [52]. The interconversion reaction between the pyranose and furanose forms of UDP-L-Ara is highlighted by a red box, and the radiolabeled sugars used for carbon flux analysis by Sharples and Fry [16] are highlighted by green boxes. Modified after Figure 1 in ref. [2]. www.sciencedirect.com
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Figure 2
Comparison between the bacterial pathway toward the synthesis of dTDP-L-rhamnose from dTDP-D-glucose and the eukaryotic pathway that converts UDP-D-glucose to UDP-L-rhamnose. In bacteria, three separate enzymes (RmlB, RmlC and RmlD) catalyze the sequential reactions that are required for the formation of dTDP-L-rhamnose. As an example for the organization of the dTDP-L-rhamnose biosynthetic genes in bacteria, the relevant part of the gene cluster from Salmonella enterica serovar Typhi CT18 [53] is shown in the top part of the figure. Note that RmlA encodes a dTDP-D-glucose pyrophosphorylase (glucose-1-phosphate:dTTP deoxythymidylyltransferase) that does not appear to have a functional homolog in plants. While there is a short intergenic region between RmlD and RmlA (indicated by a thin line), the other genes are located right next to each other but translated into separate proteins. In plants, all three reactions are catalyzed by a single polypeptide that is encoded by the highly similar RHM1, RHM2 and RHM3 genes in Arabidopsis. The N-terminal domain shown in blue is functionally homologous to RmlB from bacteria whereas the C-terminal domain shown in orange combines the functions of the bacterial RmlC and RmlD proteins. A short spacer region separates the two domains. The UER1 gene of Arabidopsis encodes a functional 3,5-epimerase-4-reductase but lacks the 4,6-dehydratase domain of the RHM proteins. The 3,5-epimerase-4reductase domain of the plant coding regions contain a single intron which is not shown for clarity purposes.
[35,36]. The biochemical assays on recombinant RHM2 confirmed the predicted enzymatic activities and revealed strong inhibition of the 4,6-dehydratase domain by UDP-Xyl and UDP-Rha [34]. While the inhibitory action of UDP-Xyl may be because of its structural similarity to UDP-Glc, the inhibition by UDP-Rha is likely to serve regulatory purposes by reducing the production of UDP-Rha if sufficient amounts of this product are available. UDP-Rha can only be used by glycosyltransferases for the synthesis of low-molecular-weight glycoconjugates and cell wall polysaccharides, and is apparently not metabolized by other biochemical pathways. This makes it imperative that an effective feedback inhibition mechanism for its synthesis is in place. The proposed 3,5-epimerase-4-reductase function of UER1 Current Opinion in Plant Biology 2008, 11:236–243
was verified by Watt et al. [37] by enzyme assays on the recombinant enzyme. Its precise function is unclear but it is tempting to speculate that it serves to metabolize small amounts of the 4-keto-6-deoxy intermediate that could be released by the 4,6-dehydratase domain of the RHM proteins. Two allelic mutants of the RHM1 gene were recently isolated during a screen for suppressors of the lrx1 mutation [38]. LRX1 belongs to a family of wall-resident leucine-rich repeat (LRR) proteins that contain an extensin-like domain at the carboxy-terminus [39]. Mutations in the LRX1 gene lead to the production of highly deformed root hairs that may collapse or form branchlike structures [40]. In an attempt to elucidate the mechanism underlying the lrx phenotype, Diet et al. [38] screened EMS-mutagenized lrx1 plants for restoration of the wild type phenotype, and positionally cloned a mutant gene (rol1) that acted as a suppressor of the lrx1 phenotype. ROL1 is allelic to RHM1, and the mutations in this gene turned out to cause loss of function of the encoded UDP-Rha synthase [38]. Although the rhamnose content in roots from rol1 plants remained unchanged, immunoreactivity with an RG-I-specific antibody and the RG-II content of cell wall material were decreased [38]. These results are consistent with a defect in a rhamnose-biosynthetic enzyme but the precise mechanism of suppression of the lrx1 phenotype by mutations in the RHM1 gene remains to be established.
The origin of arabinofuranosyl residues in cell wall polysaccharides With few exceptions, L-arabinose residues in plant cell wall components are in their furanose rather than the thermodynamically more stable pyranose form [41]; however, the biosynthesis of nucleotide-bound Ara residues occurs via 4-epimerization of UDP-Xyl with retention of the pyranose form [42]. This begs the question at what step in the synthesis of arabinose-containing polysaccharides the pyranose form is converted to the furanose form. It has been proposed that arabinosyltransferases carry out this ring contraction [43] or that a nucleotide sugar mutase interconverts UDP-Arap and UDP-Araf. The latter hypothesis is supported by the observation that an enzyme preparation from mung bean uses UDP-Araf as the substrate for arabinosyltransfer reactions [44]. The most likely candidates for this hypothetical mutase are potential plant homologs to known UDP-Galp mutases from pathogenic microorganisms that catalyze the interconversion between UDP-Galp and UDP-Galf [45] and that are also capable to interconvert UDP-Arap and UDP-Araf in vitro [46]. Since no obvious homologs to these flavoproteins could be found in the Arabidopsis or rice genome sequences, Konishi et al. [47] purified the mutase activity from rice seedlings to near homogeneity, and used amino acid sequence information to identify the corresponding gene. It turned out to be a member of a www.sciencedirect.com
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small gene family encoding the previously enigmatic reversibly glycosylated proteins (RGPs). This class of enzymes covalently binds the sugar moiety from a large variety of nucleotide sugars but the bound sugar moiety can easily be displaced by the monosaccharide moiety of another nucleotide sugar [48,49]. RGPs are soluble cytosolic proteins but have a tendency to associate with the endomembrane system, specifically the trans-Golgi [50]. Simultaneous disruption of two of the five RGP isoforms in Arabidopsis severely affects pollen development, and double mutants in RGP1 and RGP2 could not be obtained [51]. This is consistent with the notion that the Araf residues are indispensable for the assembly of a functional cell wall because they are major components of many cell wall polysaccharides and arabinogalactan-proteins.
Conclusions and perspectives Most of the photosynthate produced by higher plants is funneled through nucleotide sugar interconversion pathways to ultimately find its way into plant cell walls. During the last few years, new and interesting information has become available on those enzymatic steps that are irreversible in vivo leading to the conclusion that feedback inhibition of key enzymes plays an important role in metabolic control. Kinetic experiments on recombinant enzymes revealed substantial differences between isoforms of the same enzyme but the significance of these observations is not understood. Because several isoforms of an enzyme are often expressed in the same cell type, the formation of mixed multimers is a distinct possibility that has rarely been addressed in experiments with recombinant enzymes. Furthermore, the activity of nucleotide sugar interconversion enzymes may be influenced by unknown activator or repressor proteins. One of the main challenges for the future is to study protein-protein interactions that may be pivotal to control the carbon flux through these metabolic pathways.
Acknowledgements This work was supported by the U.S. Department of Energy (grant No. DEFG02-95ER20203) and the National Science Foundation (grant No. IBN0215535).
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10. Klinghammer M, Tenhaken R: Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls. J Exp Bot 2007, 58:3609-3621. The expression pattern of the four isoforms of UDP-Glc dehydrogenase from Arabidopsis was analyzed, and isoforms UGD2, UGD3 and UGD4 were recombinantly expressed in E. coli to determine their kinetic parameters. Although some differences in the Km values for UDPGlc and the Ki values for UDP-Xyl were noted, the functional significance of these differences remains unclear. All three enzymes were specific for UDP-Glc and preferred NAD+ as the oxidizing cofactor although NADP+ was also accepted with an approximately five-fold lower efficiency. 11. Harper AD, Bar-Peled M: Biosynthesis of UDP-xylose. Cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDPglucuronic acid decarboxylase isoforms. Plant Physiol 2002, 130:2188-2198. 12. Pattathil S, Harper AD, Bar-Peled M: Biosynthesis of UDPxylose: characterization of membrane-bound AtUXS2. Planta 2005, 221:538-548. 13. Carpita N, McCann M: The cell wall. In Biochemistry and Molecular Biology of Plants. Edited by Buchanan BB, Gruissem W, Jones RL. Rockville, MD: American Society of Plant Physiologists; 2000:52-108. 14. Liepman AH, Wilkerson CG, Keegstra K: Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases. Proc Natl Acad Sci U S A 2005, 102:2221-2226. Current Opinion in Plant Biology 2008, 11:236–243
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15. Stewart DC, Copeland L: Uridine-50 -diphosphate dehydrogenase from soybean nodules. Plant Physiol 1998, 116:349-355. 16. Sharples SC, Fry SC: Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells. Plant J 2007, 52:252-262. An elegant double-labeling experiment of suspension-cultured Arabidopsis cells with [1-3H]D-galactose and [U-14C]D-fructose was conducted to evaluate the relative contributions of known or postulated pathways toward the synthesis of UDP-GlcA, UDP-GalA and GDP-Glc. The twolabeled compounds enter nucleotide sugar interconversion pathways at widely separated points via salvage pathways (galactokinase and hexokinase, respectively), which permits an evaluation of the major routes toward the synthesis of specific nucleotide sugars. The overall conclusion of this study is that UDP-GlcA is primarily synthesized via oxidation of UDP-Glc to UDP-GlcA, UDP-GalA is primarily synthesized via 4-epimerization of UDP-GlcA, and GDP-Glc is primarily synthesized by guanylyl transfer from GTP onto glucose-1-phosphate. 17. Usadel B, Schlu¨ter U, Mølhøj M, Gipmans M, Verma R, Kossmann J, Reiter W-D, Pauly M: Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis. FEBS Lett 2004, 569:327-331. 18. Mølhøj M, Verma R, Reiter W-D: The biosynthesis of Dgalacturonate in plants. Functional cloning and characterization of a membrane-anchored UDP-Dglucuronate 4-epimerase from Arabidopsis. Plant Physiol 2004, 135:1221-1230. 19. Gu X, Bar-Peled M: The biosynthesis of UDP-galacturonic acid in plants. Functional cloning and characterization of Arabidopsis UDP-D-glucuronic acid 4-epimerase. Plant Physiol 2004, 136:4256-4264. 20. Yang Y-H, Kang Y-B, Lee K-W, Lee T-H, Park S-S, Hwang B-Y, Kima B-G: Characterization of GDP-mannose pyrophosphorylase from Escherichia coli O157:H7 EDL933 and its broad substrate specificity. J Mol Catal B: Enzymatic 2005, 37:1-8. 21. Pe´aud-Lenoe¨l C, Axelos M: The purification and properties of guanosine diphosphate glucose pyrophosphorylase of pea seedlings. Eur J Biochem 1968, 4:561-567. 22. Conklin PL, Norris SR, Wheeler GL, William EH, Smirnoff N, Last RL: Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci U S A 1999, 96:4198-4203. 23. Lukowitz W, Nickle TC, Meinke DW, Last RL, Conklin PL, Somerville CR: Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis. Proc Natl Acad Sci U S A 2001, 98:2262-2267. 24. Ka¨rko¨nen A: Biosynthesis of UDP-GlcA: via UDPGDH or myoinositol oxidation pathway? Plant Biosyst 2005, 139:46-49. 25. Lorence A, Chevone BI, Mendes P, Nessler CL: Myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol 2004, 134:1200-1205. 26. Kanter U, Usadel B, Guerineau F, Li Y, Pauly M, Tenhaken R: The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides. Planta 2005, 221:243-254. 27. McCaskill A, Turgeon R: Phloem loading in Verbascum phoeniceum L. depends on the synthesis of raffinose-family oligosaccharides. Proc Natl Acad Sci U S A 2007, 104:1961919624. 28. Tenhaken R, Thulke O: Cloning of an enzyme that synthesizes a key nucleotide sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase. Plant Physiol 1996, 112:1127-1134. 29. Oka T, Jigami Y: Reconstruction of de novo pathway for synthesis of UDP-glucuronic acid and UDP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae. FEBS J 2006, 273:2645-2657. The UGD1 isoform of UDP-Glc dehydrogenase and the AXS3 isoform of UDP-Xyl synthase from Arabidopsis were expressed in the budding yeast S. cerevisiae to generate an abundant source for UDP-Xyl, and to study Current Opinion in Plant Biology 2008, 11:236–243
the regulation of these enzymes in a host that does not normally contain them. Expression of AtUGD1 alone reduced the endogenous pool of UDP-Glc by a factor of 2 but further depletion of UDP-Glc was presumably prevented by competitive inhibition of the enzyme by the product UDP-GlcA with a Ki value of about 0.1 mM. In yeast strains expressing both AtUGD1 and AtUXS3 the pool of UDP-Glc remained unchanged presumably because of strong competitive inhibition of UDP-Glc dehydrogenase by UDP-Xyl with a Ki value of about 5 mM. These results suggest that feedback inhibition of plant nucleotide sugar interconversion enzymes is sufficient to stabilize the pool size of nucleotide sugars even if the reactions are highly exergonic and the end products are not further metabolized. 30. Dong C, Beis K, Giraud MF, Blankenfeldt W, Allard S, Major LL, Kerr ID, Whitfield C, Naismith JH: A structural perspective on the enzymes that convert dTDP-D-glucose into dTDP-L-rhamnose. Biochem Soc Trans 2003, 31:532-536. 31. Giraud MF, Naismith JH: The rhamnose pathway. Curr Opin Struct Biol 2000, 10:687-696. 32. Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfield C, Coplin D, Kido N, Klena J, Maskell D, Raetz CR et al.: Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol 1996, 4:495-503. 33. Kamsteeg J, Van Brederode J, Van Nigtevecht G: The formation of UDP-L-rhamnose from UDP-D-glucose by an enzyme preparation of red campion (Silene dioica (L) Clairv) leaves. FEBS Lett 1978, 91:281-284. 34. Oka T, Nemoto T, Jigami Y: Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDPD-glucose to UDP-L-rhamnose conversion. J Biol Chem 2007, 282:5389-5403. The three predicted isoforms of UDP-Rha synthase from Arabidopsis (RHM1, RHM2 and RHM3) were heterologously expressed in S. cerevisiae and shown to have the expected enzymatic activity in vitro. This biochemical study focuses on isoform RHM2, which contains missense mutations in both alleles of the mucilage modified4 (mum4) mutants. The authors demonstrate that the N-terminal domain of RHM2 acts as a UDP-Glc 4,6-dehydratase, and that the C-terminal domain acts as a 3,5-epimerase-4-reductase that converts the reaction intermediate into UDP-Rha. Enzyme assays on RHM2 variants that contain the amino substitutions in MUM4-1 and MUM4-2 did not reveal any activity. This provides biochemical evidence that the seed coat mucilage phenotype of mum4 plants is caused by defects in a rhamnosebiosynthetic gene. 35. Usadel B, Kuschinsky AM, Rosso MG, Eckermann N, Pauly M: RHM2 is involved in mucilage pectin biosynthesis and is required for the development of the seed coat in Arabidopsis. Plant Physiol 2004, 134:286-295. 36. Western TL, Young DS, Dean GH, Tan WL, Samuels AL, Haughn GW: MUCILAGE-MODIFIED4 encodes a putative pectin biosynthetic enzyme developmentally regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis seed coat. Plant Physiol 2004, 134:296-306. 37. Watt G, Leoff C, Harper AD, Bar-Peled M: A bifunctional 3,5epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis. Plant Physiol 2004, 134:1337-1346. 38. Diet A, Link B, Seifert GJ, Schellenberg B, Wagner U, Pauly M, Reiter W-D, Ringli C: The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase. Plant Cell 2006, 18:1630-1641. A mutant screen was conducted to identify suppressors of the abnormal root hair morphology found in Arabidopsis plants that carry mutations in the cell wall protein LRX1. This protein consists of a leucine-rich repeat fused to a domain that resembles hydroxyproline-rich glycoproteins (HRGPs, also referred to as extensins). The suppressor locus rol1 (repressor of lrx1) turned out to have mutations in isoform RHM1 of UDP-Rha synthase that abolish enzymatic activity in vitro. Although the rol1 mutations were found to cause changes in rhamnogalacturonans I and II, the molecular mechanism underlying the suppressor phenotype remains to be determined. 39. Ringli C: The role of extracellular LRR-extensin (LRX) proteins in cell wall formation. Plant Biosyst 2005, 139:32-35. 40. Baumberger N, Ringli C, Keller B: The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required for root hair www.sciencedirect.com
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morphogenesis in Arabidopsis thaliana. Genes Dev 2001, 15:1128-1139. 41. O’Neill MA, York WS: The composition and structure of plant primary cell walls. In The Plant Cell Wall. Edited by Rose JKC. Oxford: Blackwell; 2003:1-54. 42. Burget EG, Verma R, Mølhøj M, Reiter WD: The biosynthesis of Larabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis. Plant Cell 2003, 15:523-531. 43. Fry SC, Northcote DH: Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides. Plant Physiol 1983, 73:1055-1061. 44. Konishi T, Ono H, Ohnishi-Kameyama M, Kaneko S, Ishii T: Identification of a mung bean arabinofuranosyltransferase that transfers arabinofuranosyl residues onto (1,5)-linked a-Larabino-oligosaccharides. Plant Physiol 2006, 141:1098-1105. 45. Beverley SM, Owens KL, Showalter M, Griffith CL, Doering DL, Jones VC, McNeil MR: Eukaryotic UDP-galactopyranose mutase (GLF gene) in microbial and metazoal pathogens. Eukaryot Cell 2005, 4:1147-1154. 46. Zhang Q, Liu H-W: Chemical synthesis of UDP-b-Larabinofuranose and its turnover to UDP-b-Larabinopyranose by UDP-galactopyranose mutase. Bioorg Med Chem Lett 2001, 11:145-149. 47. Konishi T, Takeda T, Miyazaki Y, Ohnishi-Kameyama M, Hayashi T, O’Neill MA, Ishii T: A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose. Glycobiology 2007, 17:345-354. Arabinose residues in plant cell wall material exist predominantly in their furanose form although this configuration is thermodynamically less stable than the pyranose form generated by the 4-epimerization of
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UDP-D-xylopyranose. This raises the question whether most arabinosyltransferases can catalyze the necessary ring contraction, or whether an unknown mutase catalyzes an interconversion between UDP-L-Arap and UDP-L-Araf. The authors addressed this issue by purifying UDP-L-Arap mutase activity from rice seedlings and cloning the corresponding gene. The mutase activity turned out to be a property of reversibly glycosylated proteins (RGPs) for which the biological function had not previously been established. 48. Dhugga KS, Ulvskov P, Gallagher SR, Ray PM: Plant polypeptides reversibly glycosylated by UDP-glucose – possible components of Golgi b-glucan synthase in pea cells. J Biol Chem 1991, 266:21977-21984. 49. Delgado IJ, Wang Z, de Rocher A, Keegstra K, Raikhel N: Cloning and characterization of AtRGP1—a reversibly autoglycosylated Arabidopsis protein implicated in cell wall biosynthesis. Plant Physiol 1998, 116:1339-1349. 50. Dhugga KS, Tiwari SC, Ray PM: A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: purification, gene cloning, and trans-Golgi localization. Proc Natl Acad Sci U S A 1997, 94:7679-7684. 51. Drakakaki G, Zabotina O, Delgado I, Robert S, Keegstra K, Raikhel N: Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development. Plant Physiol 2006, 142:1480-1492. 52. Li Y, Smith C, Corke F, Zheng L, Merali Z, Ryden P, Derbyshire P, Waldron K, Bevan MW: Signaling from an altered cell wall to the nucleus mediates sugar-responsive growth and development in Arabidopsis thaliana. Plant Cell 2007, 19:2500-2515. 53. Parkhill J, Dougan G, James KD, Thomson NR, Pickard D, Wain J, Churcher C, Mungall KL, Bentley SD, Holden MT et al.: Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 2001, 413:848-852.
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Biochemical genetics of nucleotide sugar interconversion reactions Wolf-Dieter Reiter During the past few years, substantial progress has been made to understand the enzymology and regulation of nucleotide sugar interconversion reactions that are irreversible in vivo on thermodynamic grounds. Feedback inhibition of enzymes by metabolic end products appears to be a common theme but some experimental results on recombinant enzymes are difficult to interpret. Using a combination of metabolic flux analysis, enzyme assays, and bioinformatics approaches, the significance of several proposed alternate pathways has been clarified. Expression of plant nucleotide sugar interconversion enzymes in yeast has become a promising approach to understand metabolic regulation and produce valuable compounds. In a major advance for the understanding of the synthesis of arabinosylated cell wall polysaccharides, reversibly glycosylated proteins turned out to act as mutases that interconvert the pyranose and furanose forms of UDP-L-arabinose. Addresses Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, USA Corresponding author: Reiter, Wolf-Dieter ([email protected])
Current Opinion in Plant Biology 2008, 11:236–243 This review comes from a themed issue on Physiology and metabolism Edited by Ken Keegstra and Markus Pauly
1369-5266/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2008.03.009
Introduction Most of the carbon that is photosynthetically fixed by plants is incorporated into cell wall material where it remains throughout the plant’s life cycle. This makes plant cell wall polysaccharides the major source of renewable biomass that can be used for the generation of ethanol via fermentation processes [1]. Because naturally occurring cell wall material is highly recalcitrant to enzymatic degradation, there is great interest in exploring possibilities to improve its deconstruction via transgenic approaches. Because glycosyltransferases in cell wall synthesis use nucleotide sugars as monosaccharide donors [2–5], modifications in the abundance of these precursors via transgenic approaches are likely to cause changes in cell wall composition that may improve the digestibility and abundance of cell wall polymers. Most genes encodCurrent Opinion in Plant Biology 2008, 11:236–243
ing nucleotide sugar interconversion enzymes have been cloned and characterized via biochemical, genetic and bioinformatics approaches, and the reader is referred to the review by Seifert [3] for coverage of the literature up to early 2004. The current review will focus on novel developments regarding carbon fluxes through nucleotide sugar interconversion pathways and the discovery of new genes.
Flux of carbon from primary photosynthesis products to cell wall material Carbon fixation in the chloroplasts leads to the formation of fructose 6-phosphate as the first phosphorylated hexose from which virtually all building blocks for the synthesis of cell wall material are derived (Figure 1). The combined action of phosphosugar isomerases and mutases yield glucose-1-phosphate and mannose-1-phosphate that are activated via cytosolic pyrophosphorylases to yield the nucleotide sugars UDP-D-glucose (UDP-Glc) and GDPD-mannose (GDP-Man) [2]. Virtually all cell wall precursors are derived from these two nucleotide sugars via a series of mechanistically related reactions that are catalyzed by 4-epimerases, 3,5-epimerases, 4-reductases, 4,6dehydratases, dehydrogenases, and decarboxylases [2,3]. With the exception of the nucleotide sugar dehydrogenases that act on carbon 6 of the sugar moiety, all of these enzymes initiate the catalytic cycle via oxidation of carbon 4 to a keto group with the help of an NAD(P)+ cofactor that is transiently reduced to NAD(P)H, and subsequently re-oxidized to its original state. The bestknown enzyme in this group is UDP-Glc 4-epimerase, which occurs in most organisms to either synthesize or degrade D-galactose. Extensive studies on the genetics and the biochemical properties of UDP-Glc 4-epimerase isoforms from Arabidopsis [6,7] and barley [8] have recently been published, and the reader is referred to these papers for detailed information. All of the epimerization reactions are freely reversible but the conversion of UDP-Glc to UDP-D-glucuronate (UDP-GlcA) [9,10] and the subsequent decarboxylation of this nucleotide sugar to UDP-D-xylose (UDP-Xyl) [11,12] are too exergonic to be reversible under in vivo conditions. This is an important issue when considering the regulation of carbon flux, in particular because sugar moieties derived from UDP-GlcA are abundant components of cell wall polymers including both pectins and hemicelluloses. For instance, D-galacturonate (GalA) is the most abundant monosaccharide in pectic cell wall material, and D-xylose (Xyl) is the predominant monosaccharide in xylans (Table 1). Like the formation of www.sciencedirect.com
Biochemical genetics of nucleotide sugar interconversion reactions Reiter 237
Table 1 UDP- and GDP-sugars, and the main cell wall components into which their monosaccharide moieties are incorporated Nucleotide sugar
Polysaccharide(s)
UDP-a-D-glucose UDP-a-D-galactose UDP-a-D-glucuronate UDP-a-D-galacturonate UDP-a-D-xylose UDP-a-D-apiose UDP-b-L-arabinose UDP-b-L-rhamnose GDP-a-D-glucose GDP-a-D-mannose GDP-b-L-galactose GDP-b-L-fucose
Cellulose (b-1 ! 4), callose (b-1 ! 3), mixed-linked glucans (b-1 ! 3 and b-1 ! 4), xyloglucan (b-1 ! 4) Rhamnogalacturonan-I and arabinogalactan-proteins (b-1 ! 3, b-1 ! 4, b-1 ! 6), xyloglucan (b-1 ! 2) Glucurono(arabino)xylans (a-1 ! 2), rhamnogalacturonan-II (b-1 ! 4) Homogalacturonan (a-1 ! 4), rhamnogalacturonan-I (a-1 ! 2), rhamnogalacturonan-II (a-1 ! 2, a-1 ! 3, a-1 ! 4) Xylans (b-1 ! 4), xyloglucan (a-1 ! 6) Rhamnogalacturonan-II (b-1 ! 2) Rhamnogalacturonan-I and arabinogalactan-proteins (a-1 ! 3, a-1 ! 5), (glucurono)arabinoxylans (a-1 ! 2, a-1 ! 3) Rhamnogalacturonan-I (a-1 ! 4), rhamnogalacturonan-II (b-1 ! 3, a-1 ! 2, a-1 ! 3, a-1 ! 5) Glucomannans (b-1 ! 4) (Gluco)mannans (b-1 ! 4) Rhamnogalacturonan-II (a-1 ! 2) Rhamnogalacturonan-II (a-1 ! 2, a-1 ! 4), xyloglucan (a-1 ! 2)
The type(s) of linkages that connect a specific sugar to other parts of the respective polysaccharides are shown in parentheses.
UDP-GlcA, the synthesis of UDP-L-rhamnose (UDPRha) from UDP-Glc and the conversion of GDP-Man to GDP-L-fucose (GDP-Fuc) are irreversible reactions under in vivo conditions, and the respective biosynthetic pathways are expected to be tightly regulated. Although the pathways shown in Figure 1 are well documented, some alternate routes toward the synthesis of nucleotide sugars have been proposed. For instance, a hypothetical 2-epimerization of GDP-Man [13] would lead to the formation of GDP-Glc that is used by glucomannan synthases in vitro [14]. This pathway would be an alternative to the synthesis of GDP-Glc from glucose-1phosphate and GTP via a pyrophosphorylase. Another unresolved issue is whether at least some UDP-GalA is formed by the dehydrogenation of UDP-Gal as described by Steward and Copeland [15] instead of the well-established 4-epimerization of UDP-GlcA. Sharples and Fry [16] recently addressed these issues by feeding suspension-cultured Arabidopsis cells a mixture of tritiated galactose and 14C-labeled fructose, and following the incorporation of the radiolabels into nucleotide sugars and cell wall polymers as a function of time. Galactose and fructose enter nucleotide sugar interconversion pathways from opposite ends of a central metabolic pathway (Figure 1), which permits an evaluation of carbon fluxes through alternate pathways by determining the 3H/14C ratio in nucleotide sugars and cell wall-derived monosaccharides [16]. The experimental data indicate that GDP-Glc is primarily formed by the action of a pyrophosphorylase, and that UDP-GalA is primarily formed by 4epimerization of UDP-GlcA, a reaction that occurs within the endomembrane system [17–19]. These results do not rule out the possibility that the proposed alternate pathways operate at low capacity in the suspension-cultured cells but at a higher capacity in specific cell types of intact plants. Nonetheless, the apparent absence of UDP-Gal dehydrogenase activity is consistent with the observation that recombinant UDP-Glc dehydrogenase from soybean [9] and three of the four isoforms of UDP-Glc dehydrowww.sciencedirect.com
genase from Arabidopsis do not act on UDP-Gal (the remaining isoform UGD1 was not tested) [10]. Furthermore, the Arabidopsis genome does not contain closely related sequences that would qualify as candidate genes for UDP-Gal dehydrogenase, and no sequence entries for UDP-Gal dehydrogenase exist in the NCBI databases. All of this suggests that this enzymatic activity is absent from all major groups of organisms including plants. The controversy regarding the formation of GDP-Glc is more difficult to address via bioinformatics approaches because neither GDP-Glc pyrophosphorylase nor the hypothetical GDP-Man 2-epimerase have been cloned from plants. Some GDP-Man pyrophosphorylases such as the enzyme from a strain of Escherichia coli have been shown to synthesize GDP-Glc from glucose-1-phosphate and GTP [20], and the same may be true for the corresponding enzymes from plants. A study on partially purified GDP-Glc pyrophosphorylase from pea seedlings indicated that mannose-1-phosphate is not a substrate for this enzyme [21] but the gene encoding this enzyme remains to be cloned. It would be interesting to determine whether any of the three predicted GDP-Man pyrophosphorylases in the Arabidopsis genome accept glucose-1-phosphate as a substrate. Leaky mutations in one of the GDP-Man pyrophosphorylase isoforms cause a reduction in ascorbic acid content (vtc1) [22], and tight mutations in the same gene (cyt1) are seedling-lethal presumably because of a defect in the N-glycosylation of cellulose synthases [23]. This strongly suggests that the VTC1/CYT1 gene is the predominant GDP-Man pyrophosphorylase in Arabidopsis whereas the functions of its two homologs are unknown. The work by Sharples and Fry [16] also addresses the flux through a well-known alternate pathway toward the synthesis of UDP-GlcA, namely the oxidation of myoinositol to GlcA via myo-inositol oxygenase (MIOX) [24]. The GlcA formed in this reaction is then converted to UDP-GlcA via a salvage pathway (blue-boxed area in Current Opinion in Plant Biology 2008, 11:236–243
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Figure 1
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Biochemical genetics of nucleotide sugar interconversion reactions Reiter 239
Figure 1). In the suspension-cultured cell system, flux through this pathway appeared to be very low compared to the oxidation of UDP-Glc to UDP-GlcA but the situation may be very different in other cell types or plant species. For example, there are large differences in the expression levels of the known [25] or predicted MIOX isoforms in Arabidopsis, and in the number of MIOX-related ESTs between plant species [26]. It is unclear at this time whether the MIOX pathway makes any significant contribution toward the synthesis of cell wall material or whether it is simply involved in the turnover of myo-inositol that is primarily used for the synthesis of phospholipids or galactinol, the precursor for the production of raffinose family oligosaccharides [27].
activities of UGD1 and UXS3 in Arabidopsis cells were similar to those in the transgenic yeast line, this would raise the question how plant cells maintain an adequate pool of UDP-GlcA for incorporation into cell wall material and the synthesis of UDP-GalA within the Golgi. It appears that the activity of the various cytosolic and membrane-bound UDP-Xyl synthase isoforms needs to be tightly regulated to circumvent problems. It could be argued that a high rate of transport of UDP-GlcA across the Golgi membrane would shield it from decarboxylation by cytosolic UDP-Xyl synthase activity but several isoforms of UDP-Xyl synthase are targeted to the endomembrane system [2,11] where they are expected to compete with UDP-GlcA 4-epimerases and glucuronosyltransferases for their common substrate UDP-GlcA.
A gene encoding UDP-Glc dehydrogenase was initially cloned from soybean [28] but more recent studies focused on the four-member UDP-Glc dehydrogenase gene family from Arabidopsis. Oka and Jigami [29] functionally expressed the UGD1 isoform of this enzyme in the yeast Saccharomyces cerevisiae either alone or in combination with the cytosolic UXS3 isoform of UDP-Xyl synthase from Arabidopsis. Because wild type yeast contains neither enzyme and does not utilize the respective end products, it is an ideal system to overproduce rare nucleotide sugars, and to study the regulation of enzymatic activities both in vivo and in vitro. Expression of UGD1 alone caused massive accumulation of UDP-GlcA and an approximately 50% reduction in the pool of UDPGlc. Complete conversion of UDP-Glc to UDP-GlcA was presumably prevented by product inhibition of the enzyme with a Ki value of 99 mM [29]. In yeast cells overexpressing both UDP-Glc dehydrogenase and UDPXyl synthase, the pools of UDP-Glc and UDP-Xyl were similar to each other, and no depletion of UDP-Glc was observed. This is presumably because of the strong competitive inhibition of UGD1 by UDP-Xyl with a Ki value of 4.9 mM [29]. Recombinant UDP-Glc dehydrogenase from soybean shows similar inhibition by UDPXyl with a Ki value of 10 mM [9] but the UGD2, UGD3 and UGD4 isoforms from Arabidopsis are only weakly inhibited by UDP-Xyl [10]. Yeast cells expressing both UDP-Glc dehydrogenase and UDP-Xyl synthase did not contain detectable amounts of UDP-GlcA indicating that this substrate is rapidly metabolized to UDP-Xyl. If the
The biosynthesis of UDP-L-rhamnose: consolidating three enzymes into one In prokaryotes, dTDP-D-glucose (dTDP-Glc) rather than UDP-Glc is set aside for the synthesis of activated Lrhamnose to separate the nucleotide sugar pools for central metabolic pathways from those for the synthesis of capsular polysaccharides [30,31]. The three steps in this bacterial interconversion reaction are catalyzed by three separate enzymes, a 4,6-dehydratase, a 3,5-epimerase, and a 4-reductase that are usually referred to as RmlB, RmlC and RmlD, respectively [32] (Figure 2). In plants, biochemical aspects of the analogous conversion of UDPGlc to UDP-Rha have been unclear because the enzyme was never purified to homogeneity although fractionated cell extracts have been used to synthesize UDP-Rha in vitro [33]. An evaluation of sequence similarities between the bacterial Rml proteins and the Arabidopsis genome sequence led to the proposal that there are three genes (RHM1, RHM2 and RHM3) encoding proteins where a 4,6-dehydratase domain is translationally fused to a combined 3,5-epimerase-4-reductase domain [2]. A separate coding region (UER1) is highly homologous to the 3,5epimerase-4-reductase domain of the RHM proteins but does not encompass the 4,6-dehydratase domain. To test the proposed function of the RHM proteins, Oka et al. [34] recombinantly expressed the RHM2 protein and its two predicted subdomains in yeast, and measured nucleotide sugar interconversion activities. The RHM2 isoform was chosen because mutants in this gene had been shown to be defective in the production of seed coat mucilage
(Figure 1 Legend ) Overview of the main nucleotide sugar interconversion reactions in plants, and their relationship to central pathways in carbohydrate metabolism. Sugar phosphates in central metabolic pathways are boxed in orange. Mannose-1-phosphate (upper part of orange box) is activated to GDP-D-Man, which is the precursor of GDP-sugars except for GDP-D-Glc. Glucose-1-phosphate (lower part of the orange box) is activated to UDP-D-Glc, which is converted into other UDP-sugars via branched pathways. Single arrows denote reactions that are essentially irreversible in vivo, and double arrows denote reactions that are expected to be close to equilibrium. Some of the salvage pathways for free monosaccharides are included in this figure. Note that the salvage pathway for D-GlcA connects the oxidative breakdown of myo-inositol to the formation of nucleotide sugars (blue box). Bars across arrows indicate known mutations in Arabidopsis that affect at least one isoform of an interconversion enzyme. hsr8 (high sugar response8; lower right corner) is a recently isolated mutant allele of the MUR4-encoded UDP-D-Xyl 4-epimerase [52]. The interconversion reaction between the pyranose and furanose forms of UDP-L-Ara is highlighted by a red box, and the radiolabeled sugars used for carbon flux analysis by Sharples and Fry [16] are highlighted by green boxes. Modified after Figure 1 in ref. [2]. www.sciencedirect.com
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Figure 2
Comparison between the bacterial pathway toward the synthesis of dTDP-L-rhamnose from dTDP-D-glucose and the eukaryotic pathway that converts UDP-D-glucose to UDP-L-rhamnose. In bacteria, three separate enzymes (RmlB, RmlC and RmlD) catalyze the sequential reactions that are required for the formation of dTDP-L-rhamnose. As an example for the organization of the dTDP-L-rhamnose biosynthetic genes in bacteria, the relevant part of the gene cluster from Salmonella enterica serovar Typhi CT18 [53] is shown in the top part of the figure. Note that RmlA encodes a dTDP-D-glucose pyrophosphorylase (glucose-1-phosphate:dTTP deoxythymidylyltransferase) that does not appear to have a functional homolog in plants. While there is a short intergenic region between RmlD and RmlA (indicated by a thin line), the other genes are located right next to each other but translated into separate proteins. In plants, all three reactions are catalyzed by a single polypeptide that is encoded by the highly similar RHM1, RHM2 and RHM3 genes in Arabidopsis. The N-terminal domain shown in blue is functionally homologous to RmlB from bacteria whereas the C-terminal domain shown in orange combines the functions of the bacterial RmlC and RmlD proteins. A short spacer region separates the two domains. The UER1 gene of Arabidopsis encodes a functional 3,5-epimerase-4-reductase but lacks the 4,6-dehydratase domain of the RHM proteins. The 3,5-epimerase-4reductase domain of the plant coding regions contain a single intron which is not shown for clarity purposes.
[35,36]. The biochemical assays on recombinant RHM2 confirmed the predicted enzymatic activities and revealed strong inhibition of the 4,6-dehydratase domain by UDP-Xyl and UDP-Rha [34]. While the inhibitory action of UDP-Xyl may be because of its structural similarity to UDP-Glc, the inhibition by UDP-Rha is likely to serve regulatory purposes by reducing the production of UDP-Rha if sufficient amounts of this product are available. UDP-Rha can only be used by glycosyltransferases for the synthesis of low-molecular-weight glycoconjugates and cell wall polysaccharides, and is apparently not metabolized by other biochemical pathways. This makes it imperative that an effective feedback inhibition mechanism for its synthesis is in place. The proposed 3,5-epimerase-4-reductase function of UER1 Current Opinion in Plant Biology 2008, 11:236–243
was verified by Watt et al. [37] by enzyme assays on the recombinant enzyme. Its precise function is unclear but it is tempting to speculate that it serves to metabolize small amounts of the 4-keto-6-deoxy intermediate that could be released by the 4,6-dehydratase domain of the RHM proteins. Two allelic mutants of the RHM1 gene were recently isolated during a screen for suppressors of the lrx1 mutation [38]. LRX1 belongs to a family of wall-resident leucine-rich repeat (LRR) proteins that contain an extensin-like domain at the carboxy-terminus [39]. Mutations in the LRX1 gene lead to the production of highly deformed root hairs that may collapse or form branchlike structures [40]. In an attempt to elucidate the mechanism underlying the lrx phenotype, Diet et al. [38] screened EMS-mutagenized lrx1 plants for restoration of the wild type phenotype, and positionally cloned a mutant gene (rol1) that acted as a suppressor of the lrx1 phenotype. ROL1 is allelic to RHM1, and the mutations in this gene turned out to cause loss of function of the encoded UDP-Rha synthase [38]. Although the rhamnose content in roots from rol1 plants remained unchanged, immunoreactivity with an RG-I-specific antibody and the RG-II content of cell wall material were decreased [38]. These results are consistent with a defect in a rhamnose-biosynthetic enzyme but the precise mechanism of suppression of the lrx1 phenotype by mutations in the RHM1 gene remains to be established.
The origin of arabinofuranosyl residues in cell wall polysaccharides With few exceptions, L-arabinose residues in plant cell wall components are in their furanose rather than the thermodynamically more stable pyranose form [41]; however, the biosynthesis of nucleotide-bound Ara residues occurs via 4-epimerization of UDP-Xyl with retention of the pyranose form [42]. This begs the question at what step in the synthesis of arabinose-containing polysaccharides the pyranose form is converted to the furanose form. It has been proposed that arabinosyltransferases carry out this ring contraction [43] or that a nucleotide sugar mutase interconverts UDP-Arap and UDP-Araf. The latter hypothesis is supported by the observation that an enzyme preparation from mung bean uses UDP-Araf as the substrate for arabinosyltransfer reactions [44]. The most likely candidates for this hypothetical mutase are potential plant homologs to known UDP-Galp mutases from pathogenic microorganisms that catalyze the interconversion between UDP-Galp and UDP-Galf [45] and that are also capable to interconvert UDP-Arap and UDP-Araf in vitro [46]. Since no obvious homologs to these flavoproteins could be found in the Arabidopsis or rice genome sequences, Konishi et al. [47] purified the mutase activity from rice seedlings to near homogeneity, and used amino acid sequence information to identify the corresponding gene. It turned out to be a member of a www.sciencedirect.com
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small gene family encoding the previously enigmatic reversibly glycosylated proteins (RGPs). This class of enzymes covalently binds the sugar moiety from a large variety of nucleotide sugars but the bound sugar moiety can easily be displaced by the monosaccharide moiety of another nucleotide sugar [48,49]. RGPs are soluble cytosolic proteins but have a tendency to associate with the endomembrane system, specifically the trans-Golgi [50]. Simultaneous disruption of two of the five RGP isoforms in Arabidopsis severely affects pollen development, and double mutants in RGP1 and RGP2 could not be obtained [51]. This is consistent with the notion that the Araf residues are indispensable for the assembly of a functional cell wall because they are major components of many cell wall polysaccharides and arabinogalactan-proteins.
Conclusions and perspectives Most of the photosynthate produced by higher plants is funneled through nucleotide sugar interconversion pathways to ultimately find its way into plant cell walls. During the last few years, new and interesting information has become available on those enzymatic steps that are irreversible in vivo leading to the conclusion that feedback inhibition of key enzymes plays an important role in metabolic control. Kinetic experiments on recombinant enzymes revealed substantial differences between isoforms of the same enzyme but the significance of these observations is not understood. Because several isoforms of an enzyme are often expressed in the same cell type, the formation of mixed multimers is a distinct possibility that has rarely been addressed in experiments with recombinant enzymes. Furthermore, the activity of nucleotide sugar interconversion enzymes may be influenced by unknown activator or repressor proteins. One of the main challenges for the future is to study protein-protein interactions that may be pivotal to control the carbon flux through these metabolic pathways.
Acknowledgements This work was supported by the U.S. Department of Energy (grant No. DEFG02-95ER20203) and the National Science Foundation (grant No. IBN0215535).
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