Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth.

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.

Heterologous expression and biological characteristics of UGPases from Lactobacillus acidophilus.

Herein, two genes (LBA0625 and LBA1719) encoding UGPases (UDP-glucose pyrophosphorylase) in Lactobacillus acidophilus (L. acidophilus) were successfully transformed into Escherichia coli BL21 (DE3) to construct recombinant overexpressing strains (E-0625, E-1719) to investigate the biological characteristics of UGPase-0625 and UGPase-1719. The active sites, polysaccharide yield, and anti-freeze-drying stress of L. acidophilus ATCC4356 were also detected. UGPase-0625 and UGPase-1719 belong to the nucleotidyltransferase of stable hydrophilic proteins; contain 300 and 294 amino acids, respectively; and have 20 conserved active sites by prediction. Αlpha-helixes and random coils were the main secondary structures, which constituted the main skeleton of UGPases. The optimal mixture for the high catalytic activity of the two UGPases included 0.5 mM UDP-Glu (uridine diphosphate glucose) and Mg2+ at 37 °C, pH 10.0. By comparing the UGPase activities of the mutant strains with the original recombinant strains, A10, L130, and L263 were determined as the active sites of UGPase-0625 (P < 0.01) and A11, L130, and L263 were determined as the active sites of UGPase-1719 (P < 0.01). In addition, UGPase overexpression could increase the production of polysaccharides and the survival rates of recombinant bacteria after freeze-drying. This is the first study to determine the enzymatic properties, active sites, and structural simulation of UGPases from L. acidophilus, providing in-depth understanding of the biological characteristics of UGPases in lactic acid bacteria.Key points• We detected the biological characteristics of UGPases encoded by LBA0625 and LBA1719.• We identified UGPase-0625 and UGPase-1719 active sites.• UGPase overexpression elevates polysaccharide levels and post-freeze-drying survival.

UDP-glucose pyrophosphorylase: Isolation, purification and characterization from developing thermotolerant wheat (Triticum aestivum) grains.

UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9) activity was determined in four different thermotolerant varieties of wheat viz. WH-1021, PBW-373, Raj-3765 and DBW-16. The specific activity of UGPase was found to be highest at 21 days after anthesis (DAA) in the variety WH-1021 which has been developed by Haryana Agricultural University, Hisar (Haryana, India). Hence, crude extract prepared from immature grains (21 days after anthesis) of WH-1021 was used for purification of UGPase using standard protein purification techniques which exploit differences in protein properties viz. ammonium sulphate fractionation (based on solubility differences), DEAE-ion exchange chromatography (based on charge differences) and molecular sieving through Sephadex G-100 gel (based on molecular mass differences). Near homogeneous enzyme preparation with molecular mass of 82 kDa and subunit molecular weight of 39 kDa was obtained. The purified enzyme had thermostability upto 50 °C. Kinetic studies revealed that the enzyme followed Michaelis Menten kinetics with Km value of 0.9 mM and 1.66 mM for UDP and PPi, respectively. Physico-chemical and kinetic characterization suggested that the enzyme UGPase from WH-1021 is a homodimer which has adapted to high temperature stress and that lower availability of substrates and high Km values may be responsible for reduced starch synthesis/grain yield.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

Oligomerization, membrane association, and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

Engineering a Robust UDP-Glucose Pyrophosphorylase for Enhanced Biocatalytic Synthesis via ProteinMPNN and Ancestral Sequence Reconstruction.

UDP-glucose is a key metabolite in carbohydrate metabolism and plays a vital role in glycosyl transfer reactions. Its significance spans across the food and agricultural industries. This study focuses on UDP-glucose synthesis via multienzyme catalysis using dextrin, incorporating UTP production and ATP regeneration modules to reduce costs. To address thermal stability limitations of the key UDP-glucose pyrophosphorylase (UGP), a deep learning-based protein sequence design approach and ancestral sequence reconstruction are employed to engineer a thermally stable UGP variant. The engineered UGP variant is significantly 500-fold more thermally stable at 60 °C and has a half-life of 49.8 h compared to the wild-type enzyme. MD simulations and umbrella sampling calculations provide insights into the mechanism behind the enhanced thermal stability. Experimental validation demonstrates that the engineered UGP variant can produce 52.6 mM UDP-glucose within 6 h in an in vitro cascade reaction. This study offers practical insights for efficient UDP-glucose synthesis methods.

Unlocking Industrial Potential: Phase-Transition Coimmobilization of Multienzyme Systems for High-Efficiency Uridine Diphosphate Galactose Production.

Transitioning from batch to continuous industrial production often improves the economic returns and production efficiency. Immobilization is a critical strategy that can facilitate this shift. This study refined the previously established method for synthesizing uridine diphosphate galactose (UDP-Gal) by employing thermophilic enzymes. Three thermophilic enzymes (galactokinase, uridine diphosphate glucose pyrophosphorylase, and inorganic pyrophosphatase) were coimmobilized on the pH-responsive carrier Eudragit S-100, promoting enzyme recovery and reuse while their industrial potential was assessed. The coimmobilization system efficiently catalyzed UDP-Gal production, yielding 13.69 mM in 1.5 h, attaining a UTP conversion rate of 91.2% and a space-time yield (STY) of 5.16 g/L/h. Moreover, the system exhibited exceptional reproducibility, retaining 58.9% of its initial activity after five cycles. This research highlighted promising prospects for coimmobilization in industrial synthesis and proposed a novel methodology for enhancing UDP-Gal production in the industry. In addition, the phase-transition property of Eudragit S-100 paves the way for further exploration with the one-pot synthesis of poorly soluble galactosides.

Octamerization is essential for enzymatic function of human UDP-glucose pyrophosphorylase.

Uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central position in carbohydrate metabolism in all kingdoms of life, since its product uridine diphosphate-glucose (UDP-glucose) is essential in a number of anabolic and catabolic pathways and is a precursor for other sugar nucleotides. Its significance as a virulence factor in protists and bacteria has given momentum to the search for species-specific inhibitors. These attempts are, however, hampered by high structural conservation of the active site architecture. A feature that discriminates UGPs of different species is the quaternary organization. While UGPs in protists are monomers, di- and tetrameric forms exist in bacteria, and crystal structures obtained for the enzyme from yeast and human identified octameric UGPs. These octamers are formed by contacts between highly conserved amino acids in the C-terminal ß-helix. Still under debate is the question whether octamerization is required for the functionality of the human enzyme. Here, we used single amino acid replacements in the C-terminal ß-helix to interrogate the impact of highly conserved residues on octamer formation and functional activity of human UGP (hUGP). Replacements were guided by the sequence of Arabidopsis thaliana UGP, known to be active as a monomer. Correlating the data obtained in blue native PAGE, size exclusion chromatography and enzymatic activity testing, we prove that the octamer is the active enzyme form. This new insight into structure-function relationships in hUGP does not only improve the understanding of the catalysis of this important enzyme, but in addition broadens the basis for studies aimed at designing drugs that selectively inhibit UGPs from pathogens.

The crystal structure of human UDP-glucose pyrophosphorylase reveals a latch effect that influences enzymatic activity.

UGPase (UDP-glucose pyrophosphorylase) is highly conserved among eukaryotes. UGPase reversibly catalyses the formation of UDP-glucose and is critical in carbohydrate metabolism. Previous studies have mainly focused on the UGPases from plants, fungi and parasites, and indicate that the regulatory mechanisms responsible for the enzyme activity vary among different organisms. In the present study, the crystal structure of hUGPase (human UGPase) was determined and shown to form octamers through end-to-end and side-by-side interactions. The observed latch loop in hUGPase differs distinctly from yUGPase (yeast UGPase), which could explain why hUGPase and yUGPase possess different enzymatic activities. Mutagenesis studies showed that both dissociation of octamers and mutations of the latch loop can significantly affect the UGPase activity. Moreover, this latch effect is also evolutionarily meaningful in UGPase from different species.

A Chimeric UDP-glucose pyrophosphorylase produced by protein engineering exhibits sensitivity to allosteric regulators.

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.

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.

Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region ("NB loop"). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called "I-loop" region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high Km with PPi (up to 33 mM), whereas Ncut deletions had greatly increased Km with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar Km values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.

Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase.

The protozoan parasite Trypanosoma brucei is the causative agent of the cattle disease Nagana and human African sleeping sickness. Glycoproteins play key roles in the parasite's survival and infectivity, and the de novo biosyntheses of the sugar nucleotides UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine, and GDP-fucose have been shown to be essential for their growth. The only route to UDP-Gal in T. brucei is through the epimerization of UDP-glucose (UDP-Glc) by UDP-Glc 4'-epimerase. UDP-Glc is also the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum and is the presumed donor for the synthesis of base J (ß-D-glucosylhydroxymethyluracil), a rare deoxynucleotide found in telomere-proximal DNA in the bloodstream form of T. brucei. Considering that UDP-Glc plays such a central role in carbohydrate metabolism, we decided to characterize UDP-Glc biosynthesis in T. brucei. We identified and characterized the parasite UDP-glucose pyrophosphorylase (TbUGP), responsible for the formation of UDP-Glc from glucose-1-phosphate and UTP, and localized the enzyme to the peroxisome-like glycosome organelles of the parasite. Recombinant TbUGP was shown to be enzymatically active and specific for glucose-1-phosphate. The high-resolution crystal structure was also solved, providing a framework for the design of potential inhibitors against the parasite enzyme.

Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae.

The capsular polysaccharide is the major virulence factor of Streptococcus pneumoniae. Previously, we identified and cloned a region from the S. pneumoniae chromosome specific for the production of type 3 capsular polysaccharide. Now, by sequencing the region and characterizing mutations genetically and in an in vitro capsule synthesis assay, we have assigned putative functions to the products of the type-specific genes. Using DNA from the right end of the region in mapping studies, we have obtained further evidence indicating that the capsule genes of each serotype are contained in a gene cassette located adjacent to this region. We have cloned the region flanking the left end of the cassette from the type 3 chromosome and have found that it is repeated in the S. pneumoniae chromosome. The DNA sequence and hybridization data suggest a model for recombination of the capsule gene cassettes that not only describes the replacement of capsule genes, but also suggests an explanation for binary capsule type formation, and the creation of novel capsule types.

Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10-30% lower than that of UGPase-2, depending on direction of the reaction, whereas its K(m) values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03-0.14 mM vs. 0.07-0.36 mM, respectively). Both proteins were "true" UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.

Crystal structure of UDP-glucose pyrophosphorylase from Yersinia pestis, a potential therapeutic target against plague.

Yersinia pestis, the causative agent of bubonic plague, is one of the most lethal pathogens in recorded human history. Today, the concern is the possible misuse of Y. pestis as an agent in bioweapons and bioterrorism. Current therapies for the treatment of plague include the use of a small number of antibiotics, but clinical cases of antibiotic resistance have been reported in some areas of the world. Therefore, the discovery of new drugs is required to combat potential Y. pestis infection. Here, the crystal structure of the Y. pestis UDP-glucose pyrophosphorylase (UGP), a metabolic enzyme implicated in the survival of Y. pestis in mouse macrophages, is described at 2.17 Šresolution. The structure provides a foundation that may enable the rational design of inhibitors and open new avenues for the development of antiplague therapeutics.

Structure and dynamics of UDP-glucose pyrophosphorylase from Arabidopsis thaliana with bound UDP-glucose and UTP.

The structure of the UDP-glucose pyrophosphorylase encoded by Arabidopsis thaliana gene At3g03250 has been solved to a nominal resolution of 1.86 Angstroms. In addition, the structure has been solved in the presence of the substrates/products UTP and UDP-glucose to nominal resolutions of 1.64 Angstroms and 1.85 Angstroms. The three structures revealed a catalytic domain similar to that of other nucleotidyl-glucose pyrophosphorylases with a carboxy-terminal beta-helix domain in a unique orientation. Conformational changes are observed between the native and substrate-bound complexes. The nucleotide-binding loop and the carboxy-terminal domain, including the suspected catalytically important Lys360, move in and out of the active site in a concerted fashion. TLS refinement was employed initially to model conformational heterogeneity in the UDP-glucose complex followed by the use of multiconformer refinement for the entire molecule. Normal mode analysis generated atomic displacement predictions in good agreement in magnitude and direction with the observed conformational changes and anisotropic displacement parameters generated by TLS refinement. The structures and the observed dynamic changes provide insight into the ordered mechanism of this enzyme and previously described oligomerization effects on catalytic activity.

Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase.

Tuberculosis, which is caused by Mycobacterium tuberculosis, remains to be a global health problem. The thick and complex cell envelope has been implicated in many aspects of the pathogenicity of M. tuberculosis. M. tuberculosis UDP-glucose pyrophosphorylase (UGP, coded by galU, Rv0993) is involved in cell envelope precursor synthesis. UGP catalyzes the reversible formation of UDP-glucose and inorganic pyrophosphate from UTP and glucose 1-phosphate (Glc-l-P). Bacterial UGPs are completely unrelated to their eukaryotic counterparts. This enzyme is recognized as a virulence factor in several bacterial species and is conserved among mycobacterial species, which makes it a good target for mycobacterial pathogenicity research. The recombinant M. tuberculosis UGP (rMtUGP) was purified in Escherichia coli and found to be stable and catalytically active. The effects of pH, temperature and Mg2+ on enzyme activity were characterized. In addition, subcellular localization studies revealed that most of M. tuberculosis UGP protein was located in the cell wall. The purification and characterization of M. tuberculosis UGP may help to decipher the pathogenicity of M. tuberculosis.

Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase.

Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.

Crystal structure and insights into the oligomeric state of UDP-glucose pyrophosphorylase from sugarcane.

UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of UDP-glucose. In sugarcane, UDP-glucose is a branch-point in the carbon channelling into other carbohydrates, such as sucrose and cellulose, which are the major factors for sugarcane productivity. In most plants, UGPase has been described to be enzymatically active in the monomeric form, while in human and yeast, homo-octamers represent the active form of the protein. Here, we present the crystal structure of UGPase from sugarcane (ScUGPase-1) at resolution of 2.0 Å. The crystals of ScUGPase-1 reveal the presence of two molecules in the asymmetric unit and the multi-angle light scattering analysis shows that ScUGPase-1 forms a mixture of species ranging from monomers to larger oligomers in solution, suggesting similarities with the orthologs from yeast and human.