UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.

INTRODUCTION

The flux through nucleotide sugar interconversion reactions is one of the main steps controlling the amount of cell wall polysaccharides in plants [1,2]. Nucleotide sugars are principally generated through de novo pathways, in which they are formed from a starting substrate such as UDP-Glc (UDP-glucose) or GDP-Man (GDP-mannose) by successive interconversion reactions. Generation of nucleotide sugars also occurs through salvage pathways, where free monosaccharides released during the metabolism of glycoconjugates are converted into nucleotide sugars via monosaccharide 1-phosphates [1,3]. The importance of the levels of nucleotide sugars has been demonstrated in Arabidopsis mutants with defects in nucleotide sugar synthesis. For example, the mur1 mutation, which abolishes the first step in the conversion of GDP-Man into GDP-L-fucose by a GDP-Man 4,6-dehydratase (EC 4.2.1.47) in the de novo pathway [4], causes replacement of L-fucosyl residues by L-galactosyl residues in the pectic component rhamnogalacturonan-II. This leads to a reduction in boron-mediated dimer formation, causing dwarf phenotypes [5]. The rhd1/reb1 mutant, in which a gene for UGE (UDP-Glc 4-epimerase) (EC 5.1.3.2), AtUGE4, is mutated, develops swollen trichoblasts and shows growth inhibition of roots. This is possibly caused by a lack of galactosyl residues in cell wall polysaccharides such as AGPs (arabinogalactan-proteins) and xyloglucan [69]. A mutation in the MUR4 gene encoding a Golgi-localized UDP-Xyl (UDP-xylose) 4-epimerase (EC 5.1.3.5) results in a decreased level (50%) of Ara (L-arabinose) in cell wall polysaccharides from leaves, possibly because of the reduced supply of UDP-Ara, which is required as the substrate for L-arabinosyltransferases in the Golgi apparatus [10,11].

Vascular plants generate UDP-Ara through both de novo and salvage pathways. In the salvage pathway, free Ara is first converted into Ara 1-P (Ara 1-phosphate) by the action of L-arabinokinase (EC 2.7.1.46) [12], and then into UDP-Ara by a UDP-sugar pyrophosphorylase (EC 2.7.7.64) using UTP as the nucleotide donor [3]. The salvage reactions are presumed to occur in the cytosol. On the other hand, the de novo pathway for UDP-Ara generation is composed of reactions occurring in both the Golgi apparatus and the cytosol. UDP-Glc is first converted into UDP-GlcA (UDP-glucuronic acid) by the action of UDP-Glc 6-dehydrogenase (EC 1.1.1.22) [13] in the cytosol. UDP-GlcA is then imported into the Golgi lumen where it undergoes decarboxylation at C-6 of the GlcA moiety to produce UDP-Xyl by UDP-GlcA decarboxylase (synonymous with UDP-Xyl synthase, EC 4.1.1.35) [14,15]. UDP-Ara is then formed from UDP-Xyl through epimerization of the hydroxy group at C-4 of the xylose moiety, which is catalysed by a membrane-anchored UDP-Xyl 4-epimerase in the Golgi apparatus [11]. A separate pool of UDP-Xyl is generated by cytosolic UDP-GlcA decarboxylases [14,15], which would then have to be transported into the Golgi. Although the mur4 mutation significantly reduces the level of UDP-Xyl 4-epimerase activity in the microsomal fraction, it does not affect the level in the soluble fraction [10]. This suggests that plants have a pathway to generate UDP-Ara from UDP-Xyl in the cytosol which is independent of that in the Golgi apparatus, and raises the question of which kind of protein catalyses the interconversion between UDP-Ara and UDP-Xyl in the cytosol.

Although the Arabidopsis genome contains hundreds of genes predicted to encode glycosyltransferases [16], their topological features, localization and mechanisms for the incorporation of monosaccharides from nucleotide sugars into polysaccharides are mostly unknown. In regard to topology, it is known that β-(1→3),(1→4)-glucan synthase utilizes UDP-Glc at the cytosolic side of the Golgi membrane [17], whereas the catalytic side of galactan-specific β-(1→4)-galactosyltransferase and pectin-specific α-(1→4)-galacturonosyltransferase faces the Golgi lumen [18,19]. It seems that for efficient synthesis of cell wall polysaccharides, vascular plants have evolved biosynthetic pathways for nucleotide sugars in the cytosol as well as in the Golgi apparatus with transporters mediating exchange between these compartments.

In the present study, a soluble protein encoded by a predicted UGE gene is shown to have UDP-Xyl 4-epimerase activity in pea (Pisum sativum L.) sprouts. Using a recombinant enzyme expressed in Escherichia coli, UDP-Xyl 4-epimerase and UGE activity of the protein was confirmed. On the basis of the properties of the enzyme, we propose that it accounts for at least some of the UDP-Xyl 4-epimerase activity in the cytosol.

EXPERIMENTAL

Sequence alignment and phylogenetic analysis

The alignment and phylogenetic analysis of amino acid sequences of UGEs were performed using the ClustalW program. Accession numbers for the clones are as follows: AnUGE1 from Aspergillus niger, XP_001401007; AoUGE1 from Aspergillus oryzae RIB40, XP_001827449; AtUGE1 from Arabidopsis thaliana, At1g12780; AtUGE2 from A. thaliana, At4g23920; AtUGE3 from A. thaliana, At1g63180; AtUGE4 from A. thaliana, At1g64440; AtUGE5 from A. thaliana, At4g10960; BcUGE1 from Bacillus cereus, ZP_01180393; BsUGE1 from Bacillus subtilis, P55180; ClUGE1 from dog (Canis lupus subsp. familiaris), XP_544499; CrUGE1 from Chlamydomonas reinhardtii, XP_001698706; CtUGE1 from guar (Cyamopsis tetragonoloba), O65781; DdUGE1 from Dictyostelium discoideum, XP_643834; DpUGE1 from Drosophila pseudoobscura, XP_001352806; DrUGE1 from zebrafish (Danio rerio), NP_001035389; GgUGE1 from red jungle fowl (Gallus gallus), XP_417833; HsUGE1 from human (Homo sapiens), Q14376; HvUGE1 from barley (Hordeum vulgare), AAX49504; HvUGE2 from barley, AAX49505; HvUGE3 from barley, AAX49503; MmGalE from house mouse (Mus musculus), NP_848476; MtUGE1 from Medicago truncatula, ACJ85116; MtUGE2 from M. truncatula, ACJ84690; MxdUGE1 from apple (Malus x domestica), BAF51705; NcUGE1 from Neurospora crassa, NCU04442.3; OlUGE1 from Ostreococcus lucimarinus, XP_001419325; OsUGE1 from rice (Oryza sativa), Os05g0595100; OsUGE2 from rice, Os08g0374800; OsUGE3 from rice, Os09g0526700; OsUGE4 from rice, Os09g0323000; OtUGE1 from Ostreococcus tauri, CAL54894; PpUGE1 from Physcomitrella patens subsp. patens, XP_001768301; PpUGE2 from P. patens subsp. patens, XP_001777464; PpUGE3 from P. patens subsp. patens, XP_001775163; PpUGE4 from P. patens subsp. patens, XP_001751529; PpUGE5 from P. patens subsp. patens, XP_001771084; PvUGE1 from Paspalum vaginatum, BAE92559; SgUGE1 from Streptococcus gordonii, Q83WI1; StUGE45 from potato (Solanum tuberosum), AAP42567; StUGE51 from potato, AAP97493; SpUGE1, from Streptococcus pneumoniae, ZP_01825231; TcUGE1 from red flour beetle (Tribolium castaneum), XP_968616; XlUGE1 from African clawed frog (Xenopus laevis), NP_001080902; YlUGE1 from Yarrowia lipolytica, XP_504440; ZmUGE1 from Zea mays, AAP68981.

Plant materials

Pea sprouts were purchased from a local market and incubated under illumination in a growth chamber at 25 °C for 1 day before preparation of the enzyme. Pea (cv. Dun) seeds were a gift from Murakami Seed (Yokohama, Japan). For RNA preparation, pea seeds were sown on 1% agar plates and grown at 25 °C for 7 days.

UDP-sugars and other compounds

UDP-Xyl was enzymatically prepared using the method of Kobayashi et al. [14] or obtained from CarboSource (University of Georgia, Athens, GA, U.S.A.). UDP-β-L-arabinopyranose was enzymatically synthesized by recombinant UDP-sugar pyrophosphorylase using Ara 1-P and UTP as substrates [3]. UDP-Glc, UDP-Gal (UDP-galactose), UDP-GlcA, UDP-GlcNAc (UDP-N-acetylglucosamine), UTP, NAD+ and NADP+ were purchased from Sigma–Aldrich.

Enzyme assays

Activity for the pea enzymes was determined using a reaction mixture containing 50 mM Tris/HCl buffer (pH 8.6), 0.1 mM NAD+, 1 mM UDP-sugar and enzyme in a final volume of 50 μl. Different incubation temperatures, 30 and 15 °C, were used for the native and recombinant enzymes respectively. After incubation for 10 min, the reaction was terminated by dipping the mixture in a boiling water bath for 2 min. The mixture was centrifuged at 10000 g for 5 min, and 20 μl aliquots were analysed on a Shimadzu LC-10AD HPLC system equipped with a CarboPac PA-1 column (4 mm×250 mm, Dionex). The column was eluted with 50 mM sodium acetate for the initial 2 min, followed by a linear gradient (50–1000 mM, 2–40 min) and an isocratic elution with the eluent (1000 mM, 40–45 min) at a flow rate of 1 ml/min and at 35 °C [3,20]. The reaction products were monitored by absorbance at 262 nm and the amount of UDP-sugar produced was estimated from the peak area based on the curve of UDP-Glc as the calibration standard. One unit of enzyme activity is defined as the amount of enzyme converting the sugar moiety of 1 μmol of UDP-sugar into its 4-epimer per min. Elution times of standard UDP-sugars and nucleotides were: UDP-GlcA, 20.9 min; UDP-Gal, 21.5 min; UDP-Glc, 22.6 min; UDP-Ara, 23.2 min; UDP-Xyl, 23.7 min; UTP, 32.7 min; and UDP-GlcNAc, 36.0 min. The concentration of protein was determined by the method of Bradford with BSA as the standard [21].

To determine the kinetic values of rPsUGE1 (recombinant P. sativum UGE1), the enzyme activity was measured in reactions with various concentrations of UDP-sugars in the range 0.02–1.0 mM. The kinetic values were calculated using the curve-fitting program GraphPad Prism.

For the kinetic evaluation of rAtUGE1–rAtUGE5 (recombinant AtUGE1–AtUGE5), the UGE activity was monitored in a continuous assay by coupling the 4-epimerization of UDP-Gal with the oxidation of UDP-Glc to UDP-GlcA in the presence of a recombinant E. coli UDP-Glc dehydrogenase and the co-factor NAD+. rAtUGE4 was incubated with 5 mM NAD+ at room temperature overnight before the assay was conducted as this was required to obtain activity. The assay was performed in a total reaction volume of 500 μl in 1-cm-pathlength cuvettes that were charged with 100 mM glycine/NaOH (pH 8.6), 15–1000 μM UDP-Gal, 2 mM NAD+ and 40 m-units of UDP-Glc dehydrogenase (Calbiochem). The reaction was started by adding 100 ng of purified recombinant enzyme, and the net NADH production was measured by following the change in the absorbance at 340 nm for 5 min at 25 °C in a GENESYS 10 spectrophotometer (Thermo Spectronic). The rate of formation of UDP-Glc, which is one-half of the rate of formation of NADH, was determined using a molecular absorption coefficient of 6.22·103 M−1·cm−1 for NADH production. The UDP-Xyl 4-epimerase activity was monitored by an end-point assay containing 0.1–5.0 mM UDP-Xyl, 100 mM glycine/NaOH (pH 8.6) and 50 ng of purified recombinant enzyme in a final reaction volume of 50 μl. The reactions were performed at 25 °C for 10 min, and stopped by the addition of trifluoroacetic acid to a final concentration of 1.6 M. Monosaccharides obtained by hydrolysis of the nucleotide sugars were quantified by gas chromatography–MS of alditol acetates [22].

Purification of the native enzyme from pea sprouts

A crude enzyme fraction containing the native enzyme was prepared from pea sprouts using the method described by Kobayashi et al. [14]. Pea sprouts (1 kg) containing epicotyls and leaves were chopped with razor blades and homogenized with twice their weight of homogenization medium containing 100 mM phosphate buffer (pH 6.9), 1 mM DTT (dithiothreitol) and 1 mM EDTA. The homogenate was filtered through three layers of nylon mesh, and then centrifuged at 1500 g for 15 min to remove cell debris. The crude enzyme fraction was further fractionated by addition of crystalline ammonium sulfate. Proteins precipitating between 40 and 70% saturation were collected by centrifugation at 12000 g for 30 min, dissolved in 120 ml of a column buffer [20 mM phosphate buffer (pH 6.9) containing 0.5 mM DTT: buffer A], and dialysed overnight against buffer A. The dialysed sample was adsorbed on to a 2.8 cm×84 cm DEAE-Sepharose FF (GE Healthcare) column that had been equilibrated with buffer A. The column was isocratically eluted with the buffer, and individual fractions were collected. The fractions with UDP-Xyl 4-epimerase activity were brought to 80% saturation with ammonium sulfate to be concentrated. The precipitated proteins were dissolved in 10 ml of buffer A containing 0.1 M NaCl, and the resulting enzyme preparation was applied to a 2.5 cm×145 cm Sephacryl S-300 (GE Healthcare) column. The column was eluted with the same buffer, and active fractions were collected. The enzyme fractions were dialysed against 1 mM phosphate buffer (pH 6.9) containing 0.5 mM DTT, and then applied to a 0.7 cm×10.6 cm hydroxylapatite (Bio-Gel HTP, Bio-Rad Laboratories) column. The column was eluted with a linear gradient of 1–200 mM phosphate buffer (pH 6.9) containing 0.5 mM DTT. The active fractions were collected, stored at 4 °C and used to determine the properties of the native enzyme.

Peptide sequencing

The enzyme, which had been partially purified by conventional chromatography, was separated by SDS/PAGE [23], blotted on to a PVDF-Plus membrane (Osmonics) and subjected to an N-terminal amino acid sequence analysis with a protein sequencer (HP G1000A, Hewlett Packard).

cDNA cloning by RT (reverse transcription)–PCR or library screening

cDNAs encoding UGEs were cloned by RT–PCR or by screening a cDNA library. To isolate the cDNA corresponding to the mature region (Val2–Pro350) of the native PsUGE1, RT–PCR was performed using a set of specific primers, PsF-1 (5′-GGATCCGTGGCTTCGTCGCAAAAG-3′) and PsR-1 (5′-GAGCTCAAGGCTTTCCTGAGTAACC-3′) designed on the basis of the reported sequence of a putative UGE from P. sativum (PSU31544) [24]. PCR was performed with the proofreading DNA-polymerase KOD-plus (Toyobo) and the above set of primers using the single-strand cDNA as a template under the following conditions: 0.5 min of denaturation at 94 °C, 0.5 min of annealing at 55 °C and 3.0 min of extension at 68 °C, for 35 cycles.

Full-length cDNA clones for AtUGE2 and AtUGE3 were isolated by screening the λPRL2 cDNA library [25] with 32P-labelled probes that were designed on the basis of sequence information from EST (expressed sequence tag) clones 142N16T7 and ATTS506 respectively. To generate plasmid constructs for recombinant expression in E. coli, segments of full-length cDNAs for AtUGE2 and AtUGE3 were amplified by PCR using the PfuTurbo DNA polymerase (Stratagene) and the following sets of primers: UGE2-F (5′-TACCATGGCGAAGAGTGTTTTGGTTACCG-3′) and UGE2-R (5′-ATCTCGAGTGAAGAGGAGCCATTGGAG-3′); UGE3-F (5′-TACATATGGGTTCTTCTGTGGAACAG-3′) and UGE3-R (5′ATGGATCCTCAATCAAGGCTTCTTCTG-3′) (restriction sites for cloning purposes are underlined). The cDNAs for AtUGE1, AtUGE4 and AtUGE5 were isolated by RT–PCR using 50 ng of DNase I-treated total leaf RNA and the OneStep RT–PCR kit (Qiagen). RT was performed at 50 °C for 30 min, followed by PCR with HotStarTaq DNA polymerase (Qiagen) and sets of sequence-specific primers under the following conditions: 0.5 min of denaturation at 94 °C, 0.5 min of annealing at 52 °C and 1.5 min of extension at 68 °C, for 40 cycles. The sets of primers used for the RT–PCR were as follows: UGE1-F (5′-TTATCTAGATTAAGAAGGAGATATACATGGGTTCTTCTGTGGAGC-3′) and UGE1-R (5′-ATCTACTCGAGAAGCTTATTCTGGTAACCCCAT-3′); UGE4-F (5′-TATTCATATGGTTGGGAATATTCTGGTGACC-3′) and UGE4-R (5′-ATTACTCGAGTTATGTTGAGTTTGGTGAAGAAC-3′); UGE5-F (5′-TTATACCATGGCTAGAAACGTTCTAGTAAG-3′) and UGE5-R (5′-TATTACTCGAGATGAGAGTTGTCTTCAGAAGAGGAATCA-3′) (restriction sites for cloning purposes are underlined).

Expression of recombinant proteins in E. coli

The coding region for PsUGE1 was inserted between the BamHI and SacI sites of the pET32a expression vector (Novagen). The construct was designed to express the recombinant enzyme fused to thioredoxin and His6 tags at the N-terminus. The plasmid construct was transformed into the BL21(DE3) strain of E. coli (Novagen). The E. coli cells were grown at 10 °C in medium containing 1.6% (w/v) tryptone and 1.6% (w/v) yeast extract, 0.5% (w/v) NaCl, 14 mM K2HPO4 and 50 μg·ml−1 ampicillin. Expression of the recombinant protein was induced at a D600 of 0.6–1.0 by addition of IPTG (isopropyl β-D-thiogalactoside) to a final concentration of 0.5 mM. The E. coli cells were harvested 24 h after induction and lysed with 0.2% lysozyme from chicken egg. The lysate was applied to a 1.5 cm×6.0 cm chelating Sepharose FF (GE Healthcare) column. The column was washed with 50 mM Tris/HCl buffer (pH 7.5) containing 50 mM imidazole, and the bound protein was then eluted with the same buffer containing 250 mM imidazole. The purified recombinant enzyme (2.4 mg) was digested with 0.6 units of thrombin (Novagen) at 22 °C for 8 h in order to remove the fused thioredoxin and His6 tags, and then dialysed overnight against 25 mM Tris/HCl buffer (pH 7.5) containing 0.5 mM DTT. The dialysed sample was applied to a 1.4 cm×1.1 cm DEAE-Sepharose FF column equilibrated with the same buffer. The column was eluted using a step gradient of 0, 30, 60, 90, 120 and 150 mM NaCl. The purified recombinant enzyme that eluted from the column with 90 mM NaCl was examined for its purity, properties and substrate specificity.

The coding regions for AtUGE1–AtUGE3 and AtUGE5 were subcloned into the pET28a expression vector (Stratagene) in-frame with C-terminal His6 tags. All of the recombinant proteins resulting from these constructs encompass the amino acid sequence of the wild-type proteins plus the C-terminal tag LEHHHHHH. In case of AtUGE1, AtUGE2 and AtUGE5, the PCR-amplified cDNAs listed above were cloned directly into the pET28a vector using the restriction sites engineered into the respective primers. The cDNA for AtUGE3 was initially cloned into pET11a, then re-amplified with the T7 promoter primer and the oligonucleotide 5′-ATCTCGAGAGGCTTCTTCTGGAAACCCCATG-3′, which introduces a XhoI restriction site (underlined). After cleavage with XbaI and XhoI, this PCR product was cloned into pET28 cleaved with the same enzymes. The AtUGE4-coding region was subcloned into the pET28a vector in-frame with an N-terminal His6 tag. The resulting recombinant protein is predicted to encompass the entire wild-type amino acid sequence preceded by the N-terminal extension MGSSHHHHHHSSGLVPRGSH. All pET28a constructs were sequenced and transformed into the Rosetta (DE3) strain of E. coli (Novagen). The E. coli cells were grown at 37 °C in LB (Luria–Bertani) medium supplemented with 30 μg·ml−1 chloramphenicol and 50 μg·ml−1 kanamycin until a D600 of ~0.6 was attained. Expression of the recombinant protein was induced by treatment with 0.3 mM IPTG for 3 h at 30 °C. The E. coli cells were harvested, and crude lysates were prepared from frozen cell pellets. The recombinant proteins were purified using Ni-NTA (Ni2+-nitrilotriacetate) affinity columns according to the manufacturer's protocol (Qiagen). All buffers used for purification of recombinant proteins contained 1× Complete™ protease inhibitor cocktail (Roche Diagnostics).

RESULTS

Identification and purification of UDP-Xyl 4-epimerase activity from pea sprouts

We first looked for UDP-Xyl 4-epimerase activity in the soluble fraction prepared from pea sprouts, and found that the crude extract contained abundant UDP-Xyl 4-epimerase activity (approx. 0.1 unit/g of fresh weight, Table 1). The protein with UDP-Xyl 4-epimerase activity was purified by ammonium sulfate fractionation followed by several chromatographic steps. Although the enzyme was finally purified 334-fold with a yield of 7.4% (Table 1), the enzyme fraction still contained a variety of other proteins as visualized by SDS/PAGE (Figure 1A). However, the purity of the native enzyme could not be improved further by purification attempts using cation-exchange and hydrophobic chromatography (results not shown). Among the proteins in the purified fraction, the elution time of a protein with a relative molecular mass of 39 kDa (indicated with an arrow in Figure 1A) matched that of the UDP-Xyl 4-epimerase activity when each fraction eluted from the last column chromatography was subjected to enzyme assay and SDS/PAGE. This protein component was subjected to N-terminal sequence analysis. The sequence determined was VASSQKILVTGGAGFIGTHTVVQLLNNGF, which is very similar to the sequence (VASSQKILVTGSAGFIGTHTVVQLLNNGF) deduced from the cDNA encoding a putative UGE from pea (protein accession number Q43070) [24]. Among the 29 residues determined, one residue (glycine, underlined) did not match the sequence in the database. On the other hand, the cDNA (accession number AB381885) isolated from pea sprouts (cv Dun) in the present study completely matched the amino acid sequence determined for the native enzyme. The glycine residue is part of an NAD+-binding motif (GGXGXXG) [26] that is conserved for UGEs (Figure 2). The protein was designated PsUGE1 for P. sativum UGE isoform 1.

Table 1
Purification of UDP-Xyl 4-epimerase from pea sprouts

Data are based on an experiment starting with 1 kg of pea sprouts. Total activity was measured using UDP-Xyl as substrate under standard assay conditions.

Purification stage Total protein (mg) Total activity (units) Specific activity (units/mg of protein) Purification (-fold) Yield (%) 
Crude extract 5220 105 0.020 1.0 100 
40–70% ammonium sulfate 2390 56.6 0.024 1.20 54 
DEAE-Sepharose FF 123 28.7 0.233 11.7 27.3 
Sephacryl S-200 6.8 7.2 1.06 54 6.86 
Bio-Gel HTP 1.17 7.8 6.67 334 7.43 
Purification stage Total protein (mg) Total activity (units) Specific activity (units/mg of protein) Purification (-fold) Yield (%) 
Crude extract 5220 105 0.020 1.0 100 
40–70% ammonium sulfate 2390 56.6 0.024 1.20 54 
DEAE-Sepharose FF 123 28.7 0.233 11.7 27.3 
Sephacryl S-200 6.8 7.2 1.06 54 6.86 
Bio-Gel HTP 1.17 7.8 6.67 334 7.43 

SDS/PAGE of native and recombinant enzymes

Figure 1
SDS/PAGE of native and recombinant enzymes

(A) An enzyme fraction obtained after successive purification steps from pea sprouts. Lane 1, native enzyme partially purified from pea sprouts. The arrow indicates the protein band later subjected to N-terminal amino acid sequence analysis. Proteins in the gel were stained with a silver reagent. (B) rPsUGE1 at different purification steps. Lane 2, lysate of E. coli; lane 3, rPsUGE1 purified on a chelating column; lane 4, rPsUGE1 after thrombin digestion followed by purification on an ion-exchange column. The arrows indicate rPsUGE1 before and after digestion with thrombin. Proteins in the gel were stained with Coomassie Brilliant Blue R-250. The estimated molecular masses of the native and recombinant enzyme are shown in kDa. Approx. 1 μg of total protein was loaded in each lane.

Figure 1
SDS/PAGE of native and recombinant enzymes

(A) An enzyme fraction obtained after successive purification steps from pea sprouts. Lane 1, native enzyme partially purified from pea sprouts. The arrow indicates the protein band later subjected to N-terminal amino acid sequence analysis. Proteins in the gel were stained with a silver reagent. (B) rPsUGE1 at different purification steps. Lane 2, lysate of E. coli; lane 3, rPsUGE1 purified on a chelating column; lane 4, rPsUGE1 after thrombin digestion followed by purification on an ion-exchange column. The arrows indicate rPsUGE1 before and after digestion with thrombin. Proteins in the gel were stained with Coomassie Brilliant Blue R-250. The estimated molecular masses of the native and recombinant enzyme are shown in kDa. Approx. 1 μg of total protein was loaded in each lane.

Alignment of PsUGE1 with other UGEs

Figure 2
Alignment of PsUGE1 with other UGEs

Amino acid sequence deduced from the cloned PsUGE1 cDNA was aligned with those of seven other UGEs using the ClustalW program in multiple sequence alignment mode. The residues conserved between all eight UGEs are marked with asterisks, and those conserved between at least five UGEs are marked with colons. The region corresponding to the N-terminal sequence determined for the native PsUGE1 is boxed. The Gly13 residue that deviates from the sequence reported by Lake et al. [24] is shown with an arrowhead. The GGXGXXG motif involved in NAD+-binding is underlined, and a stretch of amino acid residues encompassing the catalytic site (including the completely conserved YXXXK motif) is doubly underlined. The residues corresponding to Cys307, which functions as a ‘gatekeeper’ controlling the size of the active-site cleft in HsUGE1 [27], are indicated with an arrow. Species are described in the Experimental section.

Figure 2
Alignment of PsUGE1 with other UGEs

Amino acid sequence deduced from the cloned PsUGE1 cDNA was aligned with those of seven other UGEs using the ClustalW program in multiple sequence alignment mode. The residues conserved between all eight UGEs are marked with asterisks, and those conserved between at least five UGEs are marked with colons. The region corresponding to the N-terminal sequence determined for the native PsUGE1 is boxed. The Gly13 residue that deviates from the sequence reported by Lake et al. [24] is shown with an arrowhead. The GGXGXXG motif involved in NAD+-binding is underlined, and a stretch of amino acid residues encompassing the catalytic site (including the completely conserved YXXXK motif) is doubly underlined. The residues corresponding to Cys307, which functions as a ‘gatekeeper’ controlling the size of the active-site cleft in HsUGE1 [27], are indicated with an arrow. Species are described in the Experimental section.

Relationship of PsUGE1 with other UGEs

In addition to the NAD+-binding motif, the amino acid sequence of PsUGE1 included a catalytic site highly conserved for UGEs (Figure 2). In a human UGE, HsUGE1, Cys307 has been identified as the ‘gatekeeper’ residue which controls the size of the active-site cleft and thereby enables the enzyme to act on UDP-GlcNAc [27]. This ‘gatekeeper’ residue is conserved in the sense that small amino acids such as cysteine or serine occur in the corresponding location in bacterial UGEs that also have UDP-GlcNAc 4-epimerase (EC 5.1.3.7) activity [28,29]. In contrast, in PsUGE1, the ‘gatekeeper’ residue is replaced with a bulky residue, valine, which probably explains the failure of PsUGE1 to act on UDP-GlcNAc (see below).

Phylogenetic relationships of PsUGE1 with the enzymes from other origins including mammals, insects, fungi and bacteria were analysed (Figure 3). Together with AtUGE1, AtUGE3, OsUGE3 and several other plant enzymes, PsUGE1 formed a plant subgroup apart from mammalian and bacterial enzymes. According to Rösti et al. [30], plant UGEs can be divided into two subgroups based on the similarity of amino acid sequences (designated plant UGE I and II families in Figure 3), which probably reflects a difference in their in vivo functions. Interestingly, the UGEs from the moss P. patens formed a separate group approximately equidistant from the plant UGE I and II families (Figure 3).

Relationships of PsUGE1 with other UGEs

Figure 3
Relationships of PsUGE1 with other UGEs

The phylogenetic relationships of PsUGE1 with UGEs from other origins were analysed using the ClustalW program. Enzymes shown to have high UDP-Xyl 4-epimerase activity are boxed. The percentages of bootstrap values for the respective branches are shown. The bar indicates 0.1 substitution per site. Species are described in the Experimental section.

Figure 3
Relationships of PsUGE1 with other UGEs

The phylogenetic relationships of PsUGE1 with UGEs from other origins were analysed using the ClustalW program. Enzymes shown to have high UDP-Xyl 4-epimerase activity are boxed. The percentages of bootstrap values for the respective branches are shown. The bar indicates 0.1 substitution per site. Species are described in the Experimental section.

Properties of rPsUGE1

The PsUGE1 cDNA corresponding to the mature enzyme (from Val2 to Pro350) was amplified by RT–PCR and cloned into the expression vector pET32a. The rPsUGE1 expressed in E. coli was purified by chelating chromatography and the N-terminal thioredoxin and oligohistidine tags were removed by thrombin digestion, which was followed by chromatography on an ion-exchange column (Table 2). The purified rPsUGE1 formed a single band with an apparent molecular mass of 41 kDa on SDS/PAGE (Figure 1B). The slightly higher molecular mass of the rPsUGE1 compared with that of the native enzyme (39 kDa) presumably resulted from a 34 amino acid sequence between the thrombin cleavage site and the PsUGE1 coding region inserted into the pET32a vector. The UDP-Xyl 4-epimerase activity of the purified rPsUGE1 was 16.7 units/mg of protein, which is higher than that of the native enzyme (6.7 units/mg of protein) partially purified from pea sprouts. The maximum UDP-Xyl 4-epimerase activity of rPsUGE1 occurred between pH 8.5 and 9.0 (Figure 4A). rPsUGE1 showed UGE activity that was also maximal between pH 8.5 and 9.0. The optimum temperature for both activities was 15 °C, and the enzyme lost both activities completely at temperatures higher than 40 °C (Figure 4B). The optimum temperature for rPsUGE1 (15 °C) was lower than that for the native enzyme (30 °C, results not shown). This may be a consequence of the vector-encoded peptide sequence attached to rPsUGE1. Since several enzymes acting on nucleotide sugars require metal ions such as Mg2+, Mn2+, Co2+ or Zn2+ [3,31], their effect on the UGE and UDP-Xyl 4-epimerase activities of rPsUGE1 was examined (see Supplementary Table S1 at http://www.BiochemJ.org/bj/424/bj4240169add.htm). The recombinant protein did not require any metal ion for either UGE or UDP-Xyl 4-epimerase activity. Moreover, the activities of rPsUGE1 were not diminished in the absence of NAD+ or NADP+ (results not shown). These properties of rPsUGE1 are shared by other UGEs isolated from vascular plants [32,33]. Most of the transition metal ions tested caused only a modest inhibition of enzymatic activity, whereas Hg2+ abolished enzyme function entirely (see Supplementary Table S1). The similarity of properties between the UGE and UDP-Xyl 4-epimerase activities suggests that the two activities occur at the same catalytic site.

Table 2
Purification of rPsUGE1 expressed in E. coli

The assay was carried out for UDP-Glc 4-epimerase using UDP-Gal as substrate under standard assay conditions. The activity towards UDP-Xyl is shown in parentheses. Data are based on an experiment starting with 250 ml of culture medium.

Purification stage Total protein (mg) Total activity (units) Specific activity (units/mg of protein) Purification (-fold) Yield (%) 
Lysate 28.9 483 (288) 16.7 (10.0) 1.0 (1.0) 100 (100.0) 
Ni–Sepharose FF 2.41 118 (51.0) 49.0 (21.2) 2.93 (2.12) 24.4 (17.7) 
DEAE-Sepharose FF 2.16 84 (36.0) 38.9 (16.7) 2.33 (1.67) 17.4 (12.5) 
Purification stage Total protein (mg) Total activity (units) Specific activity (units/mg of protein) Purification (-fold) Yield (%) 
Lysate 28.9 483 (288) 16.7 (10.0) 1.0 (1.0) 100 (100.0) 
Ni–Sepharose FF 2.41 118 (51.0) 49.0 (21.2) 2.93 (2.12) 24.4 (17.7) 
DEAE-Sepharose FF 2.16 84 (36.0) 38.9 (16.7) 2.33 (1.67) 17.4 (12.5) 

Effect of pH and temperature on UGE and UDP-Xyl 4-epimerase activities of rPsUGE1

Figure 4
Effect of pH and temperature on UGE and UDP-Xyl 4-epimerase activities of rPsUGE1

(A) Activity-pH curves of UGE (open symbols) and UDP-Xyl 4-epimerase (closed symbols). The enzyme activities were determined using the following buffers (50 mM) of differing pH: circle, MES/KOH; square, Tris/HCl; triangle, borate/NaOH. (B) Activity–temperature curves of UGE and UDP-Xyl 4-epimerase. Open and closed symbols indicate UGE and UDP-Xyl 4-epimerase respectively. Results are means±S.E.M. for triplicate assays.

Figure 4
Effect of pH and temperature on UGE and UDP-Xyl 4-epimerase activities of rPsUGE1

(A) Activity-pH curves of UGE (open symbols) and UDP-Xyl 4-epimerase (closed symbols). The enzyme activities were determined using the following buffers (50 mM) of differing pH: circle, MES/KOH; square, Tris/HCl; triangle, borate/NaOH. (B) Activity–temperature curves of UGE and UDP-Xyl 4-epimerase. Open and closed symbols indicate UGE and UDP-Xyl 4-epimerase respectively. Results are means±S.E.M. for triplicate assays.

Structure of the product UDP-Ara

To confirm the UDP-Xyl 4-epimerase activity of PsUGE1, we characterized the product generated from UDP-Xyl by rPsUGE1 using NMR. A large-scale reaction with rPsUGE1 followed by product purification via HPLC yielded approx. 1.2 mg of product showing the same retention time as authentic UDP-Ara. The structure of the purified product was investigated by NMR spectroscopy (see Supplementary Table S2 at http://www.BiochemJ.org/bj/424/bj4240169add.htm). The signals of the product in 1H- and 13C-NMR spectra were in accordance with those of UDP-β-L-arabinopyranose specimens synthesized either chemically [34] or enzymatically [3,20]. These results confirm that the product is UDP-β-L-arabinopyranose, which is an activated form of Ara in vascular plants.

Substrate specificity of rPsUGE1 towards UDP-sugars

The action of native PsUGE1 and rPsUGE1 on various UDP-sugars was examined. The recombinant protein exhibited the highest activity in the conversion of UDP-Gal into UDP-Glc, whereas it showed a lower activity in the reverse direction (Table 3) with a ratio of approx. 4:1 between UDP-Glc and UDP-Gal at equilibrium (Table 4). In contrast, the activity of rPsUGE1 toward UDP-Xyl was similar to that towards UDP-Ara, and nearly equal proportions of UDP-Ara and UDP-Xyl were present at equilibrium. In accordance with the prediction based on the ‘gatekeeper’ residue [27] (Figure 2), rPsUGE1 did not act on UDP-GlcNAc at all. The substrate specificity of rPsUGE1 towards UDP-sugars coincided with that of the native PsUGE1 partially purified from pea sprouts (Table 3).

Table 3
Activity of native and recombinant PsUGE1 towards UDP-sugars

The activities of the native and the recombinant enzymes towards various UDP-sugars were determined under standard assay conditions. Activities are compared with activity towards UDP-Gal and expressed as percentages.

 Enzyme activity 
 Native enzyme Recombinant enzyme 
Substrate (units/mg of protein) (%) (units/mg of protein) (%) 
UDP-Gal 19.8±0.2 100 53.3±1.0 100 
UDP-Glc 1.7±0.0 4.6±1.1 
UDP-Xyl 6.7±0.2 33 22.4±0.3 42 
UDP-Ara 6.7±0.5 31 21.1±1.3 40 
UDP-GlcA 0.0±0.0 0.0±0.0 
UDP-GlcNAc 0.0±0.0 0.0±0.0 
 Enzyme activity 
 Native enzyme Recombinant enzyme 
Substrate (units/mg of protein) (%) (units/mg of protein) (%) 
UDP-Gal 19.8±0.2 100 53.3±1.0 100 
UDP-Glc 1.7±0.0 4.6±1.1 
UDP-Xyl 6.7±0.2 33 22.4±0.3 42 
UDP-Ara 6.7±0.5 31 21.1±1.3 40 
UDP-GlcA 0.0±0.0 0.0±0.0 
UDP-GlcNAc 0.0±0.0 0.0±0.0 
Table 4
Proportions of UDP-sugars at equilibrium

The proportions of substrate and product UDP-sugars formed by the action of rPsUGE1 are estimated based on the total amount of the UDP-sugars.

Starting substrate Substrate (%) Product (%) 
UDP-Gal 19.1±0.3 80.9±0.3 
UDP-Glc 80.4±0.2 19.6±0.2 
UDP-Xyl 52.9±0.8 47.1±0.8 
UDP-Ara 48.1±0.0 51.9±0.0 
Starting substrate Substrate (%) Product (%) 
UDP-Gal 19.1±0.3 80.9±0.3 
UDP-Glc 80.4±0.2 19.6±0.2 
UDP-Xyl 52.9±0.8 47.1±0.8 
UDP-Ara 48.1±0.0 51.9±0.0 

Substrate saturation kinetics of rPsUGE1

The effect of substrate concentration on the activity of rPsUGE1 was examined for UDP-Gal, UDP-Glc, UDP-Xyl and UDP-Ara. The resulting Km and kcat values are listed in Table 5. Consistent with the substrate specificity towards UDP-sugars, the Km and kcat values of rPsUGE1 for UDP-Xyl (0.15 mM, 17 s−1) were similar to those for UDP-Ara (0.16 mM, 23 s−1). The kcat value of rPsUGE1 for UDP-Gal (64 s−1) was manifestly higher than that for UDP-Glc (9 s−1), which is roughly in line with the observed equilibrium constant between UDP-Glc and UDP-Gal (Table 4).

Table 5
Substrate saturation kinetics of rPsUGE1

To examine the effect of UDP-sugar concentration, enzyme activity was measured in reactions with various concentrations of UDP-sugars in the range 0.02–1.0 mM. The Km and kcat values were calculated using a direct-fit algorithm to the Michaelis–Menten equation.

Substrate Km (mM) kcat (s−1kcat/Km (mM−1·s−1
UDP-Gal 0.29±0.03 64 220 
UDP-Glc 0.31±0.03 28 
UDP-Xyl 0.15±0.02 17 115 
UDP-Ara 0.16±0.01 23 141 
Substrate Km (mM) kcat (s−1kcat/Km (mM−1·s−1
UDP-Gal 0.29±0.03 64 220 
UDP-Glc 0.31±0.03 28 
UDP-Xyl 0.15±0.02 17 115 
UDP-Ara 0.16±0.01 23 141 

UDP-Xyl 4-epimerase activity of other plant UGEs

As indicated by the phylogenetic tree, PsUGE1 belongs to plant UGE I family together with the Arabidopsis counterparts, AtUGE1 and AtUGE3. We also expressed AtUGE1–AtUGE5 in E. coli and determined the kinetic values of the recombinant enzymes for UDP-Gal and UDP-Xyl (Table 6). The kinetics of rAtUGEs for UDP-Gal have been reported previously [35], and the kinetic values of rAtUGEs were comparable with those reported, except for rAtUGE4 possibly because of differences in tagging and enzyme assay strategies. rAtUGE1 and rAtUGE3 exhibited both UGE and UDP-Xyl 4-epimerase activities, as did rPsUGE1. On the other hand, the UDP-Xyl 4-epimerase activity of rAtUGE2, rAtUGE4 and rAtUGE5, belonging to plant UGE II family, was weak. These results support the notion that high UDP-Xyl 4-epimerase activity is a common feature for the enzymes of the plant UGE I family (Figure 3).

Table 6
Kinetics of rAtUGEs

The effect of UDP-sugar concentration on enzyme activities of rAtUGEs was examined in reactions with various concentrations of UDP-Gal in the range 0.01–0.5 mM and UDP-Xyl in the range 0.1–1.0 mM.

Enzyme Km (mM) kcat (s−1kcat/Km (mM−1·s−1
UDP-Gal    
 rAtUGE1 0.087±0.008 103 
 rAtUGE2 0.095±0.005 55 578 
 rAtUGE3 0.068±0.003 27 397 
 rAtUGE4 0.057±0.002 20 350 
 rAtUGE5 0.15±0.007 64 435 
UDP-Xyl    
 rAtUGE1 0.43±0.02 19 45 
 rAtUGE2 0.34±0.03 0.4 1.2 
 rAtUGE3 0.32±0.05 13 40 
 rAtUGE4 0.42±0.06 0.2 0.5 
 rAtUGE5 0.16±0.02 0.5 3.1 
Enzyme Km (mM) kcat (s−1kcat/Km (mM−1·s−1
UDP-Gal    
 rAtUGE1 0.087±0.008 103 
 rAtUGE2 0.095±0.005 55 578 
 rAtUGE3 0.068±0.003 27 397 
 rAtUGE4 0.057±0.002 20 350 
 rAtUGE5 0.15±0.007 64 435 
UDP-Xyl    
 rAtUGE1 0.43±0.02 19 45 
 rAtUGE2 0.34±0.03 0.4 1.2 
 rAtUGE3 0.32±0.05 13 40 
 rAtUGE4 0.42±0.06 0.2 0.5 
 rAtUGE5 0.16±0.02 0.5 3.1 

DISCUSSION

UGE is widely distributed in living organisms, including bacteria, fungi, plants and mammals, where it plays a central role in galactose synthesis and utilization. The genome of Arabidopsis contains at least five genes for UGE, AtUGE1–AtUGE5, which can be divided into two subgroups, AtUGE1 and AtUGE3, and AtUGE2, AtUGE4 and AtUGE5, based on the similarity of their deduced amino acid sequences [30]. PsUGE1 is most similar to AtUGE1 (85% identical) among these. In the present study, we have demonstrated that PsUGE1, AtUGE1 and AtUGE3 catalyse interconversions between both UDP-Glc and UDP-Gal, and UDP-Xyl and UDP-Ara. This fact suggests that UGEs with UDP-Xyl 4-epimerase activity are conserved in vascular plants. Although PsUGE1 has significant sequence similarity to a human UGE with UDP-GlcNAc 4-epimerase activity [27,36], rPsUGE1 failed to act on UDP-GlcNAc in vitro. This can perhaps be explained by the presence of the large ‘gatekeeper’ residue, Val309, in PsUGE1. However, the recognition of UDP-GlcNAc and UDP-GalNAc may not be exclusively determined by the ‘gatekeeper’ residue. This is shown, e.g., by a barley UGE, HvUGE1, which has the same large ‘gatekeeper’ residue (Val309), but nevertheless exhibits a very low UDP-GlcNAc 4-epimerase activity of unknown biological significance [33]. It seems that enzymes belonging to the plant UGE I family, including PsUGE1, have acquired UDP-Xyl 4-epimerase activity and lost most of their UDP-GlcNAc 4-epimerase activity in the evolutionary process. The split of plant UGEs into two subgroups appears to be correlated with the evolution of vascular plants because UGEs from the moss P. patens and from green algae do not fall into either family I or II (Figure 3).

Consistent with the fact that native PsUGE1 was extracted as a soluble enzyme from pea sprouts, the analyses of the amino acid sequence using SignalP [37] and TargetP [38] clearly indicates that PsUGE1 contains neither a signal sequence for secretion nor an organelle-targeting sequence. Moreover, the analysis of subcellular localization using AtUGE1 fused to cyan fluorescent protein has revealed that AtUGE1 localizes diffusely in the cytosol [35]. Therefore it is not likely that PsUGE1 and the close homologues participate in the interconversion between UDP-Xyl and UDP-Ara in the Golgi apparatus, where membrane-anchored UDP-Xyl 4-epimerase is localized [10,11]. It is probable that UDP-Xyl formed in the cytosol by the action of cytosolic UDP-GlcA decarboxylase is the substrate for these enzymes. Indeed, a pea UDP-GlcA decarboxylase, PsUXS1, isolated from the soluble fraction of sprouts lacks a transmembrane region at the N-terminus [14]. In addition, genes encoding cytosolic UDP-GlcA decarboxylases have been found in other plants; examples include AtUXS3 in Arabidopsis [15] and OsUXS3 in rice [39]. Working in concert with PsUXS1, PsUGE1 probably constitutes the de novo pathway for UDP-Ara generation in the cytosol. A dual pathway generating UDP-Ara in the cytosol and the Golgi apparatus appears to be conserved in vascular plants because proteins belonging to UGE I family and homologues with the Golgi-localized UDP-Xyl 4-epimerase [11] are present in gymnosperms, grasses and dicotyledons.

Enzymes belonging to the plant UGE I family including PsUGE1 are also expected to be a central component of salvage pathways for free galactose and Ara that are released during the turnover of cell wall polysaccharides and AGPs [35]. Both monosaccharides are first converted into their respective 1-phosphates, then activated to UDP-sugars through the action of UDP-sugar pyrophosphorylases in the cytosol [3]. PsUGE1 and related enzymes may play an important role in converting excess UDP-Gal and UDP-Ara into UDP-Glc and UDP-Xyl respectively.

Since PsUGE1 shares significant sequence similarity with other UGEs with distinct substrate specificity towards UDP-sugars, it is likely that only a limited number of the residues of PsUGE1 are involved in the recognition of UDP-Xyl and UDP-Ara. To address the exact mechanisms of its bifunctional activity, stereochemical analysis of the three-dimensional structure of PsUGE1 would be required. The physiological importance of the cytosolic interconversion between UDP-Xyl and UDP-Ara catalysed by PsUGE1 also remains to be clarified.

Abbreviations

     
  • AGP

    arabinogalactan-protein

  •  
  • Ara

    L-arabinose

  •  
  • Ara 1-P

    Ara 1-phosphate

  •  
  • DTT

    dithiothreitol

  •  
  • GDP-Man

    GDP-mannose

  •  
  • IPTG

    isopropyl β-D-thiogalactoside

  •  
  • RT

    reverse transcription

  •  
  • UDP-Gal

    UDP-galactose

  •  
  • UDP-Glc

    UDP-glucose

  •  
  • UDP-GlcA

    UDP-glucuronic acid

  •  
  • UDP-GlcNAc

    UDP-N-acetylglucosamine

  •  
  • UDP-Xyl

    UDP-xylose

  •  
  • UGE

    UDP-Glc 4-epimerase

  •  
  • AtUGE

    Arabidopsis thaliana UGE

  •  
  • HsUGE

    Homo sapiens UGE

  •  
  • HvUGE

    Hordeum vulgare UGE

  •  
  • OsUGE

    Oyza sativa UGE

  •  
  • PsUGE

    Pisum sativum UGE

  •  
  • rAtUGE

    recombinant AtUGE

  •  
  • rPsUGE1

    recombinant PsUGE1

AUTHOR CONTRIBUTION

Toshihisa Kotake, Wolf-Dieter Reiter, Rajeev Verma and Yoichi Tsumuraya conceived the experiments. Toshihisa Kotake, Ryohei Takata, Masato Takaba, Daisuke Yamaguchi, Takahiro Orita and Satoshi Kaneko performed the identification and characterization of native and recombinant PsUGE1. Wolf-Dieter Reiter and Rajeev Verma determined the properties of recombinant AtUGEs. Koji Matsuoka and Toshihisa Kotake analysed the structure of UDP-Xyl synthesized with recombinant enzyme. Tetsuo Koyama, Wolf-Dieter Reiter and Rajeev Verma co-wrote the paper. All authors discussed the results and commented on the paper.

FUNDING

This research was supported in part by a Grant for Ground Research for Space Utilization to T. K. from the Japan Space Forum and a Grant-in-Aid for Scientific Research to T. K. [grant number 17770028] from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Work on the UGE isoforms from Arabidopsis was supported by the U.S. Department of Energy [grant number DE-FG02-95ER20203] to W.-D. R.

References

References
1
Reiter
W.-D.
Biochemical genetics of nucleotide sugar interconversion pathways
Curr. Opin. Plant Biol.
2008
, vol. 
11
 (pg. 
236
-
243
)
2
Seifert
G. J.
Nucleotide sugar interconversions and cell wall biosynthesis: how to bring the inside to the outside
Curr. Opin. Plant Biol.
2004
, vol. 
7
 (pg. 
277
-
284
)
3
Kotake
T.
Yamaguchi
D.
Ohzono
H.
Hojo
S.
Kaneko
S.
Ishida
H. K.
Tsumuraya
Y.
UDP-sugar pyrophosphorylase with broad substrate specificity toward various monosaccharide 1-phosphates from pea sprouts
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
45728
-
45736
)
4
Bonin
C. P.
Potter
I.
Vanzin
G. F.
Reiter
W.-D.
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
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
2085
-
2090
)
5
O'Neill
M. A.
Eberhard
S.
Albersheim
P.
Darvill
A. G.
Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth
Science
2001
, vol. 
294
 (pg. 
846
-
849
)
6
Schiefelbein
J. W.
Somerville
C.
Genetic control of root hair development in Arabidopsis thaliana
Plant Cell
1990
, vol. 
2
 (pg. 
235
-
243
)
7
Andéme-Onzighi
C.
Sivaguru
M.
Judy-March
J.
Baskin
T. I.
Driouich
A.
The reb1-1 mutation of Arabidopsis alters the morphology of trichoblasts, the expression of arabinogalactan-proteins and the organization of cortical microtubules
Planta
2002
, vol. 
215
 (pg. 
949
-
958
)
8
Nguema-Ona
E.
Andéme-Onzighi
C.
Aboughe-Angone
S.
Bardor
M.
Ishii
T.
Lerouge
P.
Driouich
A.
The reb1-1 mutation of Arabidopsis: effect on the structure and localization of galactose-containing cell wall polysaccharides
Plant Physiol.
2006
, vol. 
140
 (pg. 
1406
-
1417
)
9
Seifert
G. J.
Barber
C.
Wells
B.
Dolan
L.
Roberts
K.
Galactose biosynthesis in Arabidopsis: genetic evidence for substrate channeling from UDP-D-galactose into cell wall polymers
Curr. Biol.
2002
, vol. 
12
 (pg. 
1840
-
1845
)
10
Burget
E. G.
Reiter
W.-D.
The mur4 mutant of Arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose
Plant Physiol.
1999
, vol. 
121
 (pg. 
383
-
389
)
11
Burget
E. G.
Verma
R.
Mølhøj
M.
Reiter
W.-D.
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
Plant Cell
2003
, vol. 
15
 (pg. 
523
-
531
)
12
Sherson
S.
Gy
I.
Medd
J.
Schmidt
R.
Dean
C.
Kreis
M.
Lecharny
A.
Cobbett
C.
The arabinose kinase, ARA1, gene of Arabidopsis is a novel member of the galactose kinase gene family
Plant Mol. Biol.
1999
, vol. 
39
 (pg. 
1003
-
1012
)
13
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
, vol. 
112
 (pg. 
1127
-
1134
)
14
Kobayashi
M.
Nakagawa
H.
Suda
I.
Miyagawa
I.
Matoh
T.
Purification and cDNA cloning of UDP-D-glucuronate carboxy-lyase (UDP-D-xylose synthase) from pea seedlings
Plant Cell Physiol.
2002
, vol. 
43
 (pg. 
1259
-
1265
)
15
Harper
A. D.
Bar-Peled
M.
Biosynthesis of UDP-xylose: cloning and characterization of a novel Arabidopsis gene family, UXS, encoding soluble and putative membrane-bound UDP-glucuronic acid decarboxylase isoforms
Plant Physiol.
2002
, vol. 
130
 (pg. 
2188
-
2198
)
16
Egelund
J.
Skjøt
M.
Geshi
N.
Ulvskov
P.
Petersen
B. L.
A complementary bioinformatics approach to identify potential plant cell wall glycosyltransferase-encoding genes
Plant Physiol.
2004
, vol. 
136
 (pg. 
2609
-
2620
)
17
Urbanowicz
B. R.
Rayon
C.
Carpita
N. C.
Topology of the maize mixed linkage (1→3),(1→4)-β-D-glucan synthase at the Golgi membrane
Plant Physiol.
2004
, vol. 
134
 (pg. 
758
-
768
)
18
Sterling
J. D.
Quigley
H. F.
Orellana
A.
Mohnen
D.
The catalytic site of the pectin biosynthetic enzyme α-1,4-galacturonosyltransferase is located in the lumen of the Golgi
Plant Physiol.
2001
, vol. 
127
 (pg. 
360
-
371
)
19
Geshi
N.
Jørgensen
B.
Ulvskov
P.
Subcellular localization and topology of β-(1→4)galactosyltransferase that elongates β-(1→4)galactan side chains in rhamnogalacturonan I in potato
Planta
2004
, vol. 
218
 (pg. 
862
-
868
)
20
Pauly
M.
Porchia
A.
Olsen
C. E.
Nunan
K. J.
Scheller
H. V.
Enzymatic synthesis and purification of uridine diphospho-β-L-arabinopyranose, a substrate for the biosynthesis of plant polysaccharides
Anal. Biochem.
2000
, vol. 
278
 (pg. 
69
-
73
)
21
Bradford
M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
1976
, vol. 
72
 (pg. 
248
-
254
)
22
Diet
A.
Link
B.
Seifert
G. J.
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
, vol. 
18
 (pg. 
1630
-
1641
)
23
Laemmli
U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Nature
1970
, vol. 
227
 (pg. 
680
-
685
)
24
Lake
M. R.
Williamson
C. L.
Slocum
R. D.
Molecular cloning and characterization of a UDP-glucose-4-epimerase gene (galE) and its expression in pea tissues
Plant Physiol. Biochem.
1998
, vol. 
36
 (pg. 
555
-
562
)
25
Newman
T.
de Bruijn
F. J.
Green
P.
Keegstra
K.
Kende
H.
McIntosh
L.
Ohlrogge
J.
Raikhel
N.
Somerville
S.
Thomashow
M.
, et al. 
Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones
Plant Physiol.
1994
, vol. 
106
 (pg. 
1241
-
1255
)
26
Bellamacina
C. R.
The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins
FASEB J.
1996
, vol. 
10
 (pg. 
1257
-
1269
)
27
Schulz
J. M.
Watson
A. L.
Sanders
R.
Ross
K. L.
Thoden
J. B.
Holden
H. M.
Fridovich-Keil
J. L.
Determinants of function and substrate specificity in human UDP-galactose 4′-epimerase
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
32796
-
32803
)
28
Xu
D.-Q.
Thompson
J.
Cisar
J. O.
Genetic loci for coaggregation receptor polysaccharide biosynthesis in Streptococcus gordonii 38
J. Bacteriol.
2003
, vol. 
185
 (pg. 
5419
-
5430
)
29
Soldo
B.
Scotti
C.
Karamata
D.
Lazarevic
V.
The Bacillus subtilis Gne (GneA, GalE) protein can catalyse UDP-glucose as well as UDP-N-acetylglucosamine 4-epimerisation
Gene
2003
, vol. 
319
 (pg. 
65
-
69
)
30
Rösti
J.
Barton
C. J.
Albrecht
S.
Dupree
P.
Pauly
M.
Findlay
K.
Roberts
K.
Seifert
G. J.
UDP-glucose 4-epimerase isoforms UGE2 and UGE4 cooperate in providing UDP-galactose for cell wall biosynthesis and growth of Arabidopsis thaliana
Plant Cell
2007
, vol. 
19
 (pg. 
1565
-
1579
)
31
Rudick
V. L.
Weisman
R. A.
Uridine diphosphate glucose pyrophosphorylase of Acanthamoeba castellanii: purification, kinetic, and developmental studies
J. Biol. Chem.
1974
, vol. 
249
 (pg. 
7832
-
7840
)
32
Dörmann
P.
Benning
C.
Functional expression of uridine 5′-diphospho-glucose 4-epimerase (EC 5.1.3.2) from Arabidopsis thaliana in Saccharomyces cerevisiae and Escherichia coli
Arch. Biochem. Biophys.
1996
, vol. 
327
 (pg. 
27
-
34
)
33
Zhang
Q.
Hrmova
M.
Shirley
N. J.
Lahnstein
J.
Fincher
G. B.
Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.)
Biochem. J.
2006
, vol. 
394
 (pg. 
115
-
124
)
34
Ernst
C.
Klaffke
W.
Chemical synthesis of uridine diphospho-D-xylose and UDP-L-arabinose
J. Org. Chem.
2003
, vol. 
68
 (pg. 
5780
-
5783
)
35
Barber
C.
Rösti
J.
Rawat
A.
Findlay
K.
Roberts
K.
Seifert
G. J.
Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
17276
-
17285
)
36
Piller
F.
Hanlon
M. H.
Hill
R. L.
Co-purification and characterization of UDP-glucose 4-epimerase and UDP-N-acetylglucosamine 4-epimerase from porcine submaxillary glands
J. Biol. Chem.
1983
, vol. 
258
 (pg. 
10774
-
10778
)
37
Bendtsen
J. D.
Nielsen
H.
von Heijne
G.
Brunak
S.
Improved prediction of signal peptides: SignalP 3.0
J. Mol. Biol.
2004
, vol. 
340
 (pg. 
783
-
795
)
38
Emanuelsson
O.
Nielsen
H.
Brunak
S.
von Heijne
G.
Predicting subcellular localization of proteins based on their N-terminal amino acid sequence
J. Mol. Biol.
2000
, vol. 
300
 (pg. 
1005
-
1016
)
39
Suzuki
K.
Watanabe
K.
Masumura
T.
Kitamura
S.
Characterization of soluble and putative membrane-bound UDP-glucuronic acid decarboxylase (OsUXS) isoforms in rice
Arch. Biochem. Biophys.
2004
, vol. 
431
 (pg. 
169
-
177
)

Author notes

The nucleotide sequence data reported for Pisum sativum UDP-glucose 4-epimerase has been submitted to the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under accession number AB381885.

Supplementary data