A bifunctional enzyme with L-fucokinase and GDP-L-fucose pyrophosphorylase activities salvages free L-fucose in Arabidopsis

Monomeric sugars generated during the metabolism of polysaccharides, glycoproteins, and glycolipids are imported to the cytoplasm and converted to respective nucleotide sugars via monosaccharide 1-phosphates, to be reutilized as activated sugars. Because L-fucose (L-Fuc) is activated mainly in the form of GDP derivatives in seed plants, the salvage reactions for L-Fuc are expected to be independent from those for Glc, Gal, L-arabinose, and glucuronic acid, which are activated as UDP-sugars. For this study we have identified, in the genomic data base of Arabidopsis, the gene (designated AtFKGP) of a bifunctional enzyme with similarity to both L-fucokinase and GDP-L-Fuc pyrophosphorylase. Recombinant AtFKGP (rAt-FKGP) expressed in Escherichia coli showed both L-fucokinase and GDP-L-Fuc pyrophosphorylase activities, generating GDP-L-Fuc from L-Fuc, ATP, and GTP as the starting substrates. Point mutations in rAtFKGPs at either Gly(133) or Gly(830) caused loss of GDP-L-Fuc pyrophosphorylase and l-fucokinase activity, respectively. The apparent K(m) values of L-fucokinase activity of rAtFKGP for L-Fuc and ATP were 1.0 and 0.45 mm, respectively, and those of GDP-L-Fuc pyrophosphorylase activity for L-Fuc 1-phosphate and GTP were 0.052 and 0.17 mm, respectively. The expression of AtFKGP was detected in most cell types of Arabidopsis, indicating that salvage reactions for free L-Fuc catalyzed by AtFKGP occur ubiquitously in Arabidopsis. Loss-of-function mutants with tDNA insertion in AtFKGP exhibited higher accumulation of free L-Fuc in the soluble fraction than the wild-type plant. These results indicate that AtFKGP is a bifunctional enzyme with L-fucokinase and GDP-L-Fuc pyrophosphorylase activities, which salvages free L-Fuc in Arabidopsis.     

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP)

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.     

Arabidopsis UDP-sugar pyrophosphorylase: Evidence for two isoforms

Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (USP1) and 66 kDa (USP2) in crude extracts of Arabidopsis tissues. The 66 kDa isoform was not the result of proteolytic cleavage of USP1 during extraction. Trypsin digestion of bands on SDS gels corresponding to the location of the two isoforms followed by tandem mass spectrometry confirmed that USP peptides were present in both bands. Both USP isoforms were detected in the cytosol as determined by immunoblots of cellular fractions obtained by differential centrifugation. However, some USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity (UDP-GlcA→GlcA-1-P) measured in floret extracts. These results indicate that USP is the only pyrophosphorylase that utilizes UDP-GlcA as a substrate and suggest that it serves as the terminal enzyme of the myo-inositol oxidation pathway.     

Leishmania UDP-sugar Pyrophosphorylase: THE MISSING LINK IN GALACTOSE SALVAGE?

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.     

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min^-1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.     

Preparation of UDP-galacturonic acid using UDP-sugar pyrophosphorylase

UDP-galacturonic acid, the activated form of galacturonic acid (GalUA), is synthesized both de novo and by salvage pathways. The UDP-GalUA pyrophosphorylase gene involved in the salvage pathway has not been identified. Here we show that UDP-sugar pyrophosphorylase from Pisum sativum with a broad specificity has UDP-GalUA pyrophosphorylase activity. The enzyme catalyzed the formation of UDP-GalUA and pyrophosphate from GalUA 1-phosphate and UTP with an equilibrium constant value of 0.24. The recombinant UDP-sugar pyrophosphorylase had optimal pH of 6.0, and the apparent K(m) values for GalUA 1-phosphate, UTP, UDP-GalUA, and pyrophosphate were 2.27, 1.15, 0.70, and 1.26 mM, respectively. In the presence of inorganic pyrophosphatase, the recombinant enzyme produced UDP-GalUA in an 84% yield (based on the GalUA 1-phosphate substrate) on a preparative scale. Thus, this UDP-sugar pyrophosphorylase is useful for the highly efficient production of UDP-GalUA for studies on pectin biosynthesis.     

UDP-sugar pyrophosphorylase controls the activity of proceeding sugar-1-kinases enzymes

Plant cell wall synthesis requires a number of different nucleotide sugars which provide the building blocks of the different polymers. These nucleotide sugars are mainly provided by de novo synthesis but recycling pathways also contribute to the pools. The last enzyme of the recycling pathway is UDP-sugar pyrophosphorylase (USP), a single copy gene in Arabidopsis, of which a knockout is lethal for pollen development. Here we analyze the dependency between USP enzyme activity and the upstream glucuronokinase. Gene silencing of USP by miRNA cause a concomitant reduction of USP and of glucuronokinase activity presumably to prevent the accumulation of sugar-1-phosphates interfering with normal metabolism and depleting the phosphate pool of the cell.     

Structural basis for the broad substrate range of the UDP-sugar pyrophosphorylase from Leishmania major

Nucleotide sugars and the enzymes that are responsible for their synthesis are indispensable for the production of complex carbohydrates and, thus, for elaboration of a protective cellular coat for many organisms such as the protozoan parasite Leishmania. These activated sugars are synthesized de novo or derived from salvaged monosaccharides. In addition to UDP-glucose (UDP-Glc) pyrophosphorylase, which catalyzes the formation of UDP-Glc from substrates UTP and glucose-1-phosphate, Leishmania major and plants express a UDP-sugar pyrophosphorylase (USP) that exhibits broad substrate specificity in vitro. The enzyme, likely involved in monosaccharide salvage, preferentially generates UDP-Glc and UDP-galactose, but it may also activate other hexose- or pentose-1-phosphates such as galacturonic acid-1-phosphate or arabinose-1-phosphate. In order to gain insight into structural features governing the differences in substrate specificity, we determined the crystal structure of the L. major USP in the APO-, UTP-, and UDP-sugar-bound conformations. The overall tripartite structure of USP exhibits a significant structural homology to other nucleotidyldiphosphate-glucose pyrophosphorylases. The obtained USP structures reveal the structural rearrangements occurring during the stepwise binding process of the substrates. Moreover, the different product complexes explain the broad substrate specificity of USP, which is enabled by structural changes in the sugar binding region of the active site.     

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAβ1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.     

A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases

Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.     

Cloning of Glucuronokinase from Arabidopsis thaliana, the Last Missing Enzyme of the myo-Inositol Oxygenase Pathway to Nucleotide Sugars

Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a K_m of 0.7 mm. It requires ATP as phosphate donor (K_m 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.     

Arabidopsis INOSITOL TRANSPORTER4 mediates high-affinity H+ symport of myoinositol across the plasma membrane

Four genes of the Arabidopsis (Arabidopsis thaliana) monosaccharide transporter-like superfamily share significant homology with transporter genes previously identified in the common ice plant (Mesembryanthemum crystallinum), a model system for studies on salt tolerance of higher plants. These ice plant transporters had been discussed as tonoplast proteins catalyzing the inositol-dependent efflux of Na(+) ions from vacuoles. The subcellular localization and the physiological role of the homologous proteins in the glycophyte Arabidopsis were unclear. Here we describe Arabidopsis INOSITOL TRANSPORTER4 (AtINT4), the first member of this subgroup of Arabidopsis monosaccharide transporter-like transporters. Functional analyses of the protein in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes characterize this protein as a highly specific H(+) symporter for myoinositol. These activities and analyses of the subcellular localization of an AtINT4 fusion protein in Arabidopsis and tobacco (Nicotiana tabacum) reveal that AtINT4 is located in the plasma membrane. AtINT4 promoter-reporter gene plants demonstrate that AtINT4 is strongly expressed in Arabidopsis pollen and phloem companion cells. The potential physiological role of AtINT4 is discussed.     

Cloning and expression analysis of a UDP-galactose/glucose pyrophosphorylase from melon fruit provides evidence for the major metabolic pathway of galactose metabolism in raffinose oligosaccharide metabolizing plants

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by alpha-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism.