| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online March 12, 2004; 10.1104/pp.103.037192 Plant Physiology 134:1337-1346 (2004) © 2004 American Society of Plant Biologists A Bifunctional 3,5-Epimerase/4-Keto Reductase for Nucleotide-Rhamnose Synthesis in Arabidopsis1Complex Carbohydrate Research Center and Department of Plant Biology, University of Georgia, Athens, Georgia 30602–4712
L-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-  -L-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP--L-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 30°C. The apparent Km for NADPH is 90 µM and 16.9 µM for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP--L-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.
L-Rhamnose is a component of the plant cell wall pectic polysaccharides rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II; Ridley et al., 2001  ) and is also present in diverse secondary metabolites including anthocyanins, flavonoids, and triterpenoids (Das et al., 1987; Bar-Peled et al., 1991; van Setten et al., 1995; Shinozaki et al., 1996; Markham et al., 2000), in certain types of plant glycoproteins (Haruko and Haruko, 1999), and in arabinogalactan proteins (Pellerin et al., 1995). The specific enzymes that attach rhamnose to each molecule are known as rhamnosyltransferases (RhaTs). To date, only a small number of RhaTs have been studied, and those were involved in flavonoid rhamnosylation. The characterized RhaTs utilize UDP--L-rhamnose (UDP--L-Rha) as the donor substrate (Kamsteeg et al., 1978; Feingold, 1982; Bar-Peled et al., 1991), although in mung bean (Vigna radiata), both dTDP--L-rhamnose (dTDP--L-Rha) and UDP--L-Rha were reported to act as sugar donors for the rhamnosylation of flavonoids (Barber and Neufeld, 1961). We are studying the enzymes involved in the synthesis of the nucleotide-rhamnose as part of our effort to understand the synthesis of pectic polysaccharides. To date, the rhamnosylation of plant polysaccharides and glycoproteins has not been studied. Thus, the identity of the activated form(s) of rhamnose needed for the synthesis of these macromolecules is not known with certainty. The enzymes required for the synthesis of the activated form(s) of rhamnose in plants have also not been purified.
In contrast, much more is known about the synthesis of rhamnose in microorganisms. Gram-negative bacteria are known to form dTDP-
Biosynthesis of rhamnose-nucleotides in plants has only been reported in a few studies in which UDP-  -D-Glc was used as the starting material for synthesis of UDP-Rha. As in prokaryotes, UDP--D-Glc is converted to UDP-4-keto-6-deoxy-Glc by an enzyme activity similar to the bacterial rmlB (Fig. 1). Since at least some plants generate dTDP--D-Glc (Milner and Avigad, 1965; Delmer and Albersheim, 1970) in addition to UDP--D-Glc, formation of dTDP--L-Rha from dTDP--D-Glc was reported as well (for review, see Feingold, 1982). Less is known about the subsequent reactions in the pathway, and it was suggested that the 3,5-epimerization and 4-reduction is mediated by a single enzyme (Kamsteeg et al., 1978). Which nucleotide-activated form(s) of rhamnose plants utilize for various rhamnosylation pathways (glycoproteins, polysaccharides, secondary metabolites, and arabinogalactan proteins) is still unknown.
Here we report that an Arabidopsis gene encodes a protein that contains amino acid motifs that are present in both nucleotide-sugar epimerases and reductases. The recombinant Arabidopsis protein converts dTDP-4-keto-6-deoxy-Glc to a product identified as dTDP-
Isolation and Cloning of NRS/ER from Arabidopsis
The biosynthetic genes involved in the synthesis of dTDP-
In Arabidopsis, NRS/ER (AY513232; also annotated At1g63000) appears to be transcribed in leaves, roots, and flowers as judged by reverse transcription (RT)-PCR (Fig. 4 ). These expression data are consistent with information in the EST database, since multiple NRS/ER transcript entries are in cDNA libraries prepared from Arabidopsis flower buds, green siliques, shoots, leaves, and roots (http://www.ncbi.nlm.nih.gov/dbEST/), as well as callus (massively parallel signature sequencing database; http://dbixs001.dbi.udel.edu/MPSS4).
Characterization of the Enzymatic Properties of Arabidopsis NRS/ER
The coding region of NRS/ER, containing a tag at the N-terminal region with six His, was expressed in E. coli, and the recombinant protein was purified by nickel-affinity column chromatography. The purified protein migrated as a single band on SDS-PAGE with an apparent mass of approximately 35 kD (Fig. 5A
). Such a value is consistent with the molecular weight of 35,759 predicted from the amino acid sequence of the recombinant protein. Based on the similarity of the NRS/ER to the reductase, epimerase, and dehydratase (RED) family we predicted that this plant protein is an enzyme with dual function (epimerase/reductase), as suggested by Kamsteeg et al. (1978)
Since the potential substrate for NRS/ER (dTDP-4-keto-6-deoxy-Glc) is not available, we generated it using recombinant rmlB purified from E. coli (Graninger et al., 1999 ). The bacterial rmlB preferentially converts dTDP--D-Glc to dTDP-4-keto-6-deoxy-Glc (Graninger et al., 1999). After incubating recombinant rmlB with dTDP--D-Glc, the nucleotide-4-keto-6-deoxy-Glc eluted from a reverse-phase ODS2 HPLC column as a broad peak (Fig. 5B, 3), which is consistent with the results obtained by Nakano et al. (2000). The reason for this peak broadening has not been determined, although the presence of a 4-keto group may increase the hydrophobicity of the sugar nucleotide and thereby promote interaction with the column. 4-Keto sugars are likely to exist on the column as different keto-enol isomers that may also lead to peak broadening on the ODS column. Incubating dTDP-4-keto-6-deoxy-Glc with recombinant NRS/ER in the presence of NADPH resulted in the formation of a product that co-eluted with dTDP--L-Rha (Fig. 5B, 5). This major peak, which eluted from the HPLC column at 15.3 min, was collected and analyzed by 1H-NMR spectroscopy. The 1H-NMR spectrum of the product contained a doublet-of-doublet signal at 5.21 ppm (Table I) with coupling constants, 3J1'',P of 8.8 Hz and 3J1'',2'' of less than 2 Hz, diagnostic for proton H-1 of -L-Rha. The signal at 1.30 ppm is diagnostic for the H-6 methyl protons of rhamnose. All other signals in the 1H-NMR spectra of the enzymatically synthesized compound had chemical shifts and coupling constants that are similar to dTDP--L-Rha reported by Nakano et al. (2000). For example, the coupling constant between protons 3'' and 4'', and protons 4'' and 5'' are 9.3 and 9.8 Hz, respectively, indicating the trans configuration that is expected for rhamnose (see Table I). We observed only one point of chemical shift discrepancy for proton 5', on the deoxy-ribose, from the published report (Nakano et al., 2000). Our data showed a chemical shift of 4.16 ppm (Table I), whereas Nakano et al. (2000) reported a value of 4.03 ppm for H-5' on deoxy-ribose. However, our assignment for this proton is in complete agreement with the assignment reported for other dTDP sugars (Amann et al., 2001) that indicated a chemical shift of 4.15 for proton 5' on deoxy-ribose. Thus, our data provide evidence that dTDP--L-Rha is formed when the recombinant NRS/ER is incubated with dTDP-4-keto-6-deoxy-Glc.
We further characterize NRS/ER properties using dTDP-4-keto-6-deoxy-Glc as substrate. The cofactor NADPH is required for enzymatic activity of the recombinant protein, and without it no conversion to dTDP- -L-Rha is observed (Fig. 5B, 4). We found that NADH is also a hydride ion donor for the reduction of the 4-keto derivative, but only 40% of dTDP-4-keto-6-deoxy-Glc was converted to dTDP--L-Rha by NRS/ER when 1 mM NADH was used compared to 1 mM NADPH. The addition of up to 10 mM MgCl2 or CaCl2 had no discernible effect on the activity nor was NRS/ER activity inhibited by millimolar concentrations of the divalent cation chelator EDTA. In contrast to the plant epimerase/reductase, EDTA strongly inhibits the prokaryote 3,5-epimerase and 4-reductase activities, and the magnesium ion is essential for their activities (Graninger et al., 1999). The NRS/ER is stable for at least 30 months when stored as a crude extract at –20°C. However, more than 60% of NRS/ER activity is lost when the purified enzyme is subjected to repeated freezing and thawing. The recombinant NRS/ER is active between pH 4.5 and 10.5 with maximum activity between pH 5.5 and 7.5 (Fig. 6B
). Maximum enzyme activity is obtained at 30°C, and approximately 35% of the activity is retained at 45°C (Fig. 6A). Under optimal conditions enzyme activity is linear for 30 min, and, thus, kinetic analyses were performed using 20-min reactions. The apparent Km for NADPH is 90 µM and 16.9 ± 0.7 µM for dTDP-4-keto-6-deoxy-Glc.
We tested the specificity of the NRS/ER toward other nucleotide-4-keto-6-deoxy-sugars. To determine if NRS/ER can utilize GDP-4-keto-6-deoxy-Man as a substrate we first purified recombinant GDP-Man 4,6-dehydratase (GMD) as described (Sturla et al., 1997 ) and incubated GMD with GDP--D-Man. HPLC analysis was used to confirm full conversion of GDP--D-Man to GDP-4-keto-6-deoxy-Man. The product was then incubated, in control experiments, with recombinant human GDP-Man 3,5-epimerase/4-keto-reductase (Tonetti et al., 1996) that readily converted GDP-4-keto-6-deoxy-Man to GDP-Fuc as determined by HPLC. However, no conversion to GDP-Fuc was evident when NRS/ER was incubated withGDP-4-keto-6-deoxy-Man (with or without NADPH). We also tested the activity of NRS/ER toward UDP-4-keto-6-deoxy-Glc. For these experiments we used a recombinant purified rmlB (Hegeman et al., 2002). Although the recombinant rmlB has preference for dTDP--D-Glc, Hegeman et al. (2002) reported that partial conversion of UDP--D-Glc to UDP-4-keto-6-deoxy-Glc was achieved using the rmlB. Our initial trials to convert UDP--D-Glc to UDP-4-keto-6-deoxy-Glc using recombinant rmlB failed to produce a stable major product, and only trace amounts of putative UDP-4-keto-6-deoxy-Glc were produced after 60 min. Incubation of rmlB with UDP--D-Glc for longer periods of time (up to 20 h) in an attempt to achieve more conversion to UDP-4-keto-6-deoxy-Glc resulted in partial degradation of the product, preventing quantitation and characterization of UDP-4-keto-6-deoxy-Glc. However, co-incubation of UDP--D-Glc and rmlB with NRS/ER and NADPH resulted in an HPLC peak at retention time 9.3 min, which was collected. This conversion was NADPH dependent. Since the amount of product converted was low (presumably due to the low activity of rmlB toward UDP--D-Glc), we pooled the peaks from several reactions and analyzed the product by NMR. The 1H-NMR spectrum of the enzymatic product contained a doublet-of-doublet signal at 5.21 ppm (Table II) with coupling constants, 3J1'',P of 8.8 Hz and 3J1'',2'' of less than 2 Hz, diagnostic for the H-1 of -L-Rha. The signal at 1.30 ppm is diagnostic of the H-6 methyl protons of rhamnose. Other rhamnose signals in the 1H-NMR spectra of the NRS/ER-derived compound had chemical shifts and coupling constants similar to those for dTDP--L-rhamnose (Table I). The uridine proton assignments in the 1H-NMR spectra are characteristic as those uridines of other UDP-sugars (for example, UDP-Xyl; Harper and Bar-Peled, 2002). Thus, our data provide evidence that UDP--L-Rha is formed when the recombinant NRS/ER protein is incubated with UDP-4-keto-6-deoxy-Glc. We therefore named the protein NRS/ER for nucleotide rhamnose synthase/3,5-epimerase;4-reductase. Unfortunately, due to low conversion of UDP--D-Glc to UDP-4-keto-6-deoxy-Glc by rmlB, complete enzymatic characterization of NRS/ER was not possible.
UDP-Glc, dTDP-Glc, UDP-GlcA, UDP-Xyl, GDP-Man, or GDP-Fuc were not substrates for NRS/ER either in the presence or absence of NADPH.
The Arabidopsis gene NRS/ER (also annotated At1g6300) encodes an approximately 33.5-kD enzyme (NRS/ER) which, in the presence of NADPH, converts dTDP-4-keto-6-deoxy-Glc and UDP-4-keto-6-deoxy-Glc to dTDP- -L-Rha and UDP--L-Rha, respectively (Tables I and II). NRS/ER is similar to other plant enzymes involved in the formation of UDP--L-Rha (Barber and Chang, 1967; Kamsteeg et al., 1978;Bar-Peled et al., 1991) since all these enzymes have an absolute requirement and preference for NADPH. Our data also provide support for the previous suggestion that the 3,5-epimerase and 4-reductase activities in plants may be due to a single enzyme (Kamsteeg et al., 1978; see Fig. 1).
The combined 3,5-epimerase/4-reductase activities of NRS/ER toward dTDP-4-keto-6-deoxy-Glc and UDP-4-keto-6-deoxy-Glc are similar to the single bifunctional 3,5-epimerase/4-reductase enzyme involved in the formation of GDP-
The formation of dTDP-
The Arabidopsis genome contains three additional genes (At1g53000, At1g78570, and At3g14790) that each encode large proteins (approximately 670 amino acids) having two domains: an N-terminal domain (approximately 330 amino acids long) with amino acid sequence similarity to 4,6-dehydratase followed by a C-terminal domain (greater than 320 amino acids) that shares over 80% amino acid sequence identity to the functional NRS/ER described in this report. It is likely that these three Arabidopsis genes are also involved in the synthesis of activated Rha. Indeed, we have generated recombinant At1g78570 C-terminal domain in E. coli and found that the recombinant protein is able to convert dTDP-4-keto-6-deoxy-Glc to dTDP-
In this report we identified a gene product, NRS/ER, and showed that in vitro it forms both UDP-
Cloning and RT-PCR Analysis of Arabidopsis NRS/ER
Arabidopsis expressed tag cDNA databases (dbEST) were searched to identify cDNA with amino acid sequence similarity to bacterial rmlB, rmlC, and rmlD. Several ESTs (T44775, R92767, T88368, and Z26952) that showed similarity to these bacterial gene products were identified and used to design primers to obtain the corresponding Arabidopsis genes by RT-PCR. Briefly, total RNA from Arabidopsis ecotype Columbia plants was isolated using Trizol reagent (Chomczynski, 1993
For NRS/ER expression studies in Arabidopsis Columbia, total RNA from flowers, fully expanded rosette leaves and stems of 6-week-old plants, rosette leaves of 3-week-old plants, or roots of 4-week-old plants grown in liquid media as described (Bar-Peled and Raikhel, 1997
Fifteen milliliters of an overnight culture of E. coli strain BL21(DE3)pLysS (Novagen), carrying the pET28b:81.5 (NRS/ER) or control pET28b vector alone were used to inoculate 0.5 L Luria-Bertani broth supplemented with 50 µg/mL kanamycin and 30 µg/mL chloramphenicol. Cells were grown at 37°C while shaking (200 rpm) until a cell density of A600 = approximately 0.6, and then induced by addition of isopropylthio-
The substrate dTDP-4-keto-6-deoxy-glucose was generated from dTDP-
The assays for dTDP-4-keto-6-deoxy-Glc epimerase-reductase activity (final reaction volume of 75 µL) were performed in two steps. First, dTDP-4-keto-6-deoxy-Glc was produced by incubating dTDP-
UV-absorbing peaks eluting from the SAX column were collected and lyophilized to remove the ammonium formate. The residues were dissolved in water, relyophilized twice, and then exchanged twice with 99.96% D2O. Proton NMR spectroscopy was performed at 25°C on Varian Inova spectrometers (Palo Alto, CA) operating at 500 MHz and 600 MHz (Bar-Peled et al., 2001 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers 21730455, AAB88399, AAR99502, AF051236, AK071766, AY108467, AY513232, BE052407, BF011071, BF479490, BG369398, BG587952, BQ871492, CA258761, CA784305, CAD60580, CAB07093, CB292447, CB330308, CD839832, CD875945, CF119362, CF386165, CF443605, CF512199, and NC_003454.
The authors thank Professors Paul Messner (University of Vienna), Perry Frey (Department of Biochemistry, University of Wisconsin, Madison), and Michela Tonetti (University of Genova, Italy) for their generous gifts of the gene products used in this study. We thank Michael Hahn, Debra Mohnen, and Malcolm O'Neill for their comments on the manuscript. We also thank the anonymous reviewers for their valuable comments and Bob Kuzoff for assistance with phylogenetic analyses. Received December 3, 2003; returned for revision December 23, 2003; accepted December 23, 2003.
1 This work was supported in part by the U.S. Department of Agriculture (grant no. 2002–35318–12620 to M.B.-P.) and by the U.S. Department of Energy (center grant no. DE–FG05–93ER20097) for the DOE Center for Plant and Microbial Complex Carbohydrates.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037192. * Corresponding author; e-mail peled{at}ccrc.uga.edu; fax 706–542–4412.
Allard STM, Giraud MF, Whitfield C, Graninger M, Messner P, Naismith JH (2001) The crystal structure of dTDP-D-glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar typhimurium, the second enzyme in the dTDP-L-rhamnose pathway. J Mol Biol 307: 283–295[CrossRef][ISI][Medline]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Res 25: 3389–3402 Amann S, Drager G, Rupprath C, Kirschning A, Elling L (2001) (Chemo)enzymatic synthesis of dTDP-activated 2,6-dideoxysugars as building blocks of polyketide antibiotics. Carbohydr Res 335: 23–32[CrossRef][ISI][Medline]
Bar-Peled M, Griffith CL, Doering TL (2001) Functional cloning and characterization of a UDP-glucuronic acid decarboxylase: the pathogenic fungus Cryptococcus neoformans elucidates UDP-xylose synthesis. Proc Natl Acad Sci USA 98: 12003–12008 Bar-Peled M, Lewinsohn E, Fluhr R, Gressel J (1991) UDP-rhamnose-flavanone-7-O-glucoside-2''-O-rhamnosyltransferase - purification and characterization of an enzyme catalyzing the production of bitter compounds in citrus. J Biol Chem 226: 20953–20959 Bar-Peled M, Raikhel NV (1997) Characterization of AtSEC12 and AtSAR1. Proteins likely involved in endoplasmic reticulum and Golgi transport. Plant Physiol 114: 315–324[Abstract] Barber GA, Chang MT (1967) Synthesis of uridine diphosphate L-rhamnose by enzymes of Chlorella pyrenoidosa. Arch Biochem Biophys 118: 659–663[Medline] Barber GA, Neufeld EF (1961) Rhamnosyl transfer from TDP L-rhamnose catalyzed by plant enzyme. Biochem Biophys Res Commun 6: 44–48[Medline]
Becker A, Ruberg S, Kuster H, Roxlau AA, Keller M, Ivashina T, Cheng HP, Walker GC, Puhler A (1997) The 32-kilobase exp gene cluster of Rhizobium meliloti directing the biosynthesis of galactoglucan: genetic organization and properties of the encoded gene products. J Bacteriol 179: 1375–1384 Blankenfeldt W, Kerr ID, Giraud MF, McMiken HJ, Leonard G, Whitfield C, Messner P, Graninger M, Naismith JH (2002) Variation on a theme of SDR: dTDP-6-deoxy-L-lyxo-4-hexulose reductase (Rm1D) shows a new Mg2+-dependent dimerization mode. Structure 10: 773–786[Medline]
Bonin CP, Potter I, Vanzin GF, Reiter W-D (1997) The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-mannose-4,6-dehydratase catalyzing the first step in the de novo synthesis of GDP-L-fucose. Proc Natl Acad Sci USA 94: 2085–2090 Bonin CP, Reiter W-D (2000) A bifunctional epimerase-reductase acts downstream of the MUR1 gene product and completes the de novo synthesis of GDP-L-fucose in Arabidopsis. Plant J 21: 445–454[CrossRef][ISI][Medline] Chomczynski P (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15: 536–537 Das MC, Bachhawat BK, Mahato SB, Basu MK (1987) Plant glycosides in a liposomal drug-delivery system. Biochem J 247: 359–361[Medline]
Delmer DP, Albersheim P (1970) The biosynthesis of sucrose and nucleoside diphosphate glucose in Phaseolus aureus. Plant Physiol 45: 782–786 Feingold DS (1982) Aldo (and keto) hexoses and uronic acids. In FA Loewus, W Tanner, eds, Plant Carbohydrates I, Intracellular Carbohydrates, Vol 13A. Springer-Verlag, Heidelberg, pp 3–76 Giraud MF, Leonard GA, Field RA, Berlind C, Naismith JH (2000) RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase. Nat Struct Biol 7: 398–402[CrossRef][ISI][Medline] Giraud MF, Naismith JH (2000) The rhamnose pathway. Curr Opin Struct Biol 10: 687–696[CrossRef][ISI][Medline]
Graninger M, Nidetzky B, Heinrichs DE, Whitfield C, Messner P (1999) Characterization of dTDP-4-dehydrorhamnose 3,5-epimerase and dTDP-4-dehydrorhamnose reductase, required for dTDP-L-rhamnose biosynthesis in Salmonella enterica serovar Typhimurium LT2. J Biol Chem 274: 25069–25077
Harper AD, Bar-Peled M (2002) 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 130: 2188–2198 Haruko U, Haruko O (1999) Glycobiology of the plant glycoprotein epitope: structure, immunogenicity and allergenicity of plant glycotopes. Trends Glycosci Glycotech 11: 413–428 Hegeman AD, Gross JW, Frey PA (2002) Concerted and stepwise dehydration mechanisms observed in wild-type and mutated Escherichia coli dTDP-glucose 4,6-dehydratase. Biochemistry 41: 2797–2804[CrossRef][Medline] Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ (1998) Multiple sequence alignment with Clustal X. Trends Biochem Sci 23: 403–405[CrossRef][ISI][Medline] Kamsteeg J, van Brederode J, van Nigtevecht G (1978) The formation of UDP-L-rhamnose from UDP-D-glucose by an enzyme preparation of red campion (Silene dioica (L) Clairv) leaves. FEBS Lett 91: 281–284[CrossRef][Medline]
Kneidinger B, Graninger M, Adam G, Puchberger M, Kosma P, Zayni S, Messner P (2001) Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420–91T. J Biol Chem 276: 5577–5583
Koplin R, Wang G, Hotte B, Priefer UB, Puhler A (1993) A 3.9-kb DNA region of Xanthomonas campestris pv. campestris that is necessary for lipopolysaccharide production encodes a set of enzymes involved in the synthesis of dTDP-rhamnose. J Bacteriol 175: 7786–7792
Li Q, Reeves PR (2000) Genetic variation of dTDP-L-rhamnose pathway genes in Salmonella enterica. Microbiol 146: 2291–2307 Macpherson DF, Manning PA, Morona R (1994) Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb locus of Shigella flexneri. Mol Microbiol 11: 281–292[ISI][Medline] Markham KR, Gould KS, Winefield CS, Mitchell KA, Bloor SJ, Boase MR (2000) Anthocyanic vacuolar inclusions - their nature and significance in flower colouration. Phytochemistry 55: 327–336[CrossRef][ISI][Medline]
Marolda CL, Valvano MA (1995) Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster: Identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene. J Bacteriol 177: 5539–5546 Marumo K, Lindqvist L, Verma N, Weintraub A, Reeves PR, Lindberg AA (1992) Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-D-xylo-4-hexulose and thymidine diphosphate-L-rhamnose. Production using cloned gene products and separation by HPLC. Eur J Biochem 204: 539–545[ISI][Medline]
Mattila P, Rabina J, Hortling S, Helin J, Renkonen R (2000) Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae. Glycobiology 10: 1041–1047 Milner Y, Avigad G (1965) Thymidine diphosphate nucleotides as substrates in the sucrose synthetase reaction. Nature 206: 825
Menon S, Stahl M, Kumar R, Xu GY, Sullivan F (1999) Stereochemical course and steady state mechanism of the reaction catalysed by the GDP-fucose synthetase from Escherichia coli. J Biol Chem 274: 26743–26750
Moralejo P, Egan SM, Hidalgo E, Aguilar J (1993) Sequencing and characterization of a gene cluster encoding the enzymes for L-rhamnose metabolism in Escherichia coli. J Bacteriol 175: 5585–5594
Nakano Y, Suzuki N, Yoshida Y, Nezu T, Yamashita Y, Koga T (2000) Thymidine diphosphate-6-deoxy-L-lyxo-4-hexulose reductase synthesizing dTDP-6-deoxy-L-talose from Actinobacillus actinomycetemcomitans. J Biol Chem 275: 6806–6812 Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H, McIntosh L, Ohlrogge J, Raikhel N, Somerville S, Thomashow M, Retzel E, Somerville C (1994) Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol 106: 1241–1255[Abstract] Pellerin P, Vidal S, Williams P, Brillouet JM (1995) Characterization of five type II arabinogalactan-protein fractions from red wine of increasing uronic acid content. Carbohydr Res 277: 135–143[ISI][Medline] Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfield C, Coplin D, Kido N, Klena J, Maskell D, Raetz CR, Rick PD (1996) Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol 4: 495–503[CrossRef][ISI][Medline] Ridley BL, O'Neill MA, Mohnen D (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57: 929–967[CrossRef][ISI][Medline] Rizzi M, Tonetti M, Vigevani P, Sturla L, Bisso A, Flora AD, Bordo D, Bolognesi M (1998) GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily. Structure 6: 1453–1465[Medline] Shinozaki Y, Tobita T, Mizutani M, Matsuzaki T (1996) Isolation and identification of two new diterpene glycosides from Nicotiana tabacum. Biosci Biotechnol Biochem 60: 903–905[Medline] Sturla L, Bisso A, Zanardi D, Benatti U, De Flora A, Tonetti M (1997) Expression, purification and characterization of GDP-D-mannose 4,6-dehydratase from Escherichia coli. FEBS Lett 412: 126–130[CrossRef][ISI][Medline]
Sullivan K, Kumar R, Kriz R, Stahl M, Xu G-Y, Rouse J, Chang X-J, Boodhoo A, Potvin B, Cumming DA (1998) Molecular cloning of Human GDP-mannose 4,6-dehydratase and reconstructions of GDP-fucose biosynthesis in vitro. J Biol Chem 273: 8193–8202 Swofford DL (1998) PAUP. Phylogenetic Analysis Using Parsimony and Other Methods. Version 4. Sinauer Associates, Sunderland, MA
Tonetti M, Sturla L, Bisso A, Benatti U, De Flora A (1996) Synthesis of GDP-fucose by the Human FX protein. J Biol Chem 271: 27274–27279
Tonetti M, Zanardi D, Gurnon JR, Fruscione F, Armirotti A, Damonte G, Sturla L, De Flora A, Van Etten JL (2003) Paramecium bursaria Chlorella virus 1 encodes two enzymes involved in the biosynthesis of GDP-L-fucose and GDP-D-rhamnose. J Biol Chem 278: 21559–21565 van Setten DC, van de Werken G, Zomer G, Kersten GF (1995) Glycosyl compositions and structural characteristics of the potential immuno-adjuvant active saponins in the Quillaja saponaria Molina extract quil A. Rapid Commun Mass Spectrom 9: 660–666[CrossRef][Medline] Wierenga RK, Terpstra P, Hol WGJ (1986) Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol 187: 101–107[CrossRef][ISI][Medline] Zhang L, al-Hendy A, Toivanen P, Skurnik M (1993) Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. Mol Microbiol 9: 309–321[CrossRef][Medline] This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
max for thymidine and uridine were 267 nm and 261 nm, respectively. The columns were calibrated with authentic nucleotide-sugars (Sigma, St. Louis). 
