Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.     

Cd2+对三角褐指藻生长及尿苷二磷酸葡萄糖焦磷酸化酶基因表达调控的影响

为了探究Cd2+对三角褐指藻(Phaeodactylum tricornutum)生长及尿苷二磷酸葡萄糖焦磷酸化酶(UDP-glucose pyrophosphorylase, UGPase)基因表达调控的影响, 研究以不同浓度Cd2+处理三角褐指藻, 测定其生长、叶绿素荧光参数、UGP基因转录水平、UGPase活性和金藻昆布多糖含量等指标。结果表明: 低浓度(0.1 μmol/L)Cd2+促进三角褐指藻生长, UGP基因转录水平、UGPase活性和金藻昆布多糖含量分别比对照组提高了100.65%、11.99%和9.77%;而高浓度Cd2+处理组(2和5 μmol/L)各指标均显著降低, UGP基因转录水平较对照组分别下调了50.31%和60.47%, UGPase活性降低56.27%和66.72%, 金藻昆布多糖含量减少42.41%和47.30%。研究首次探究了Cd2+对三角褐指藻UGP基因表达的影响, 表明低浓度Cd2+促进三角褐指藻生长, 上调UGP基因表达, 提高金藻昆布多糖含量, 为Cd2+调控UGP基因的表达提供理论指导。     

Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major

Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential.     

Dosage suppression of the Kluyveromyces lactis zymocin by Saccharomyces cerevisiae ISR1 and UGP1

The Kluyveromyces lactis zymocin complex kills Saccharomyces cerevisiae cells in a process that involves tRNA cleavage by its tRNAse gamma-toxin subunit. In contrast to the gamma-toxin mode of action, the early steps of the zymocin response are less well characterized. Here, we present high-dosage suppressors of zymocin that encode a putative Pkc1-related kinase (ISR1) and UDP-glucose pyrophosphorylase (UGPase) (UGP1). Anti-UGPase Western blots and GAL10 - ISR1 overexpression suggest that zymocin suppression correlates with overproduction of UGPase or Isr1. As judged from protection against exo-zymocin and unaltered sensitivity to endogenous gamma-toxin, high-copy ISR1 and UGP1 operate in early, nontarget steps of the zymocin pathway. Consistent with a recent report on in vitro phosphorylation of Isr1 and UGPase by the CDK Pho85, high-copy ISR1 and UGP1 suppression of zymocin is abolished in a pho85 null mutant lacking CDK activity of Pho85. Moreover, suppression requires UGPase enzyme activity, and ISR1 overexpression also protects against CFW, a chitin-interfering poison. Our data agree with roles for UGPase in cell wall biosynthetic processes and for Isr1 in Pkc1-related cell wall integrity. In sum, high-copy ISR1 and UGP1 cells affect early steps of the zymocin response and potentially prevent the lethal K. lactis killer complex from establishing cell surface recognition and/or contact.     

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.     

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.     

转UGPase基因喜树木材化学成分与生长速率研究

分析了田间栽培条件下2年生转UGPase基因喜树与对照株的木材化学成分与生长速率。结果表明,转UGPase基因喜树综纤维素含量达到78.87%,比对照株相比提高了2.23%;纤维素含量为36.34%,与对照株相比没有明显提高;木质素含量为15.05%,较对照株降低了1.75%;两者的灰分含量均较低且无显著差异;冷、热水抽提物含量为7.62%与10.17%,分别提高了2.04%与2.13%;1% NaOH抽提物含量为27.13%,提高了1.27%。因此,就综纤维素、木质素、灰分含量而言,转UGPase基因喜树为优质纸浆材,水抽提物和1% NaOH抽提物的含量略高,在纸浆生产中需加以重视。本文还对转UGPase基因喜树与对照株的株高、基径、生物量进行了动态监测,结果表明,从5月25日到11月10日的生长季中,其株高平均增加121 cm,对照株平均仅67.8 cm,株高生长速率提高了78.47%;基径平均增加1 792 cm,对照株平均仅0.532 8 cm,提高了236.37%;地上部分生物量的积累与对照相比提高了322.61%,即转入UGPase基因使喜树生长速率显著提高。因此,虽然转UGPase基因喜树的综纤维素和纤维素含量没有明显提高,但其生长速率快,生物量增长显著,间接提高了纤维素与喜树碱的产量。因此,转基因喜树较普通喜树更符合纸浆材速生、纤维素含量高和产量高、木质素含量低的基本要求,可在生产中进一步推广。     

Structural basis for the reaction mechanism of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg²⁺-bound forms at 2.9 Å and 2.3 Å resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg²⁺ ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.     

Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae

Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGP1 are not viable. Two strategies were developed to drastically reduce the intracellular concentration of UDP-Glc in order to study the consequences of this metabolic engineering on physiology and morphology. Firstly, UGP1 was placed under the strongly regulatable THI4 promoter. This resulted in a 95% reduction of UGPase activity in the presence of thiamine. The phenotypic effects of this reduction were slightly stronger than those of glucose on the GAL10/CYC1-UGP1 gene fusion [Daran et al. (1995) Eur. J. Biochem. 230, 520–530]. A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutase genes in the ugp1 conditional strain. The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell wall interfering drugs. Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and β-glucans. Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears sufficient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the inability of yeast cells to properly form the cell wall.     

<Emphasis Type="Italic">FLOURY ENDOSPERM8</Emphasis>, encoding the UDP-glucose pyrophosphorylase 1, affects the synthesis and structure of starch in rice endosperm

Cereal opaque-kernel mutants are ideal genetic materials for studying the mechanism of starch biosynthesis and amyloplast development. Here we isolated and identified two allelic floury endosperm 8 (flo8) mutants of rice, named flo8-1 and flo8-2. In the flo8 mutant, the starch content was decreased and the normal physicochemical features of starch were altered. Map-based cloning and subsequent DNA sequencing analysis revealed a single nucleotide substitution and an 8-bp insertion occurred in UDP-glucose pyrophosphorylase 1 (Ugp1) gene in flo8-1 and flo8-2, respectively. Complementation of the flo8-1 mutant restored normal seed appearance by expressing full length coding sequence of Ugp1. RT-qPCR analysis revealed that Ugp1 was ubiquitously expressed. Mutation caused the decreased UGPase activity and affected the expression of most of genes associated with starch biosynthesis. Meanwhile, western blot and enzyme activity analyses showed the comparability of protein levels and enzyme activity of most tested starch biosynthesis related genes. Our results demonstrate that Ugp1 plays an important role for starch biosynthesis in rice endosperm.     

Analysis of the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA

The expression of the enzyme UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) from potato (Solanum tuberosum L.) was analysed with respect to sink-source interactions and potato tuber storage. The highest level of expression was found in developing tubers, the strongest sink tissue. Storage of mature tubers at low temperatures led to an increase of the steady-state level of UGPase mRNA, implicating a role of this enzyme in the process of cold-sweetening. Transgenic plants were created expressing UGPase antisensee RNA under the control of the 35S promoter of the Cauliflower Mosaic Virus with the polyadenylation signal of the octopine-synthase gene. Regenerated plants were tested for reduction of UGPase at the RNA, protein and activity levels. Plants with a 95%–96% reduction of UGPase activity in growing tubers showed no change in growth and development. Also, carbohydrate metabolism in tubers of these plants was not substantially affected, indicating that only 4% of the wild-type UGPase activity is sufficient for the enzyme to function in plant growth and development.Abbreviations cDNA copy DNA - CaMV Cauliflower Mosaic Virus - Glc1P glucose-1-phosphate - UDPGlc UDP-glucose - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - UGPase UDP-glucose pyrophosphorylase We are grateful to Dr. J.P. Spychalla (Cambridge Laboratory, Norwich, Norfolk, UK) for providing antiserum directed against the potato tuber UGPase protein. We thank J. Bergstein and B. Schäfer for photographic work, J. Dietze for plant transformation and R. Breitfeld and B. Burose for taking care of the greenhouse plants.     

Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction

Expanding the scope of stereoselectivity is of current interest in enzyme catalysis. In this study, using error-prone polymerase chain reaction (PCR), a thermostable adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) from Thermus caldophilus GK-24 has been altered to improve its catalytic activity toward enatiomeric substrates including [glucose-1-phosphate (G-1-P) + uridine triphosphate (UTP)] and [N-acetylglucosamine-1-phosphate (GlcNAc) + UTP] to produce uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine, respectively. To elucidate the amino acids responsible for catalytic activity, screening for UDP-glucose pyrophosphorylase (UGPase) and UDP-N-acetylglucosamine pyrophosphorylase (UNGPase) activities was carried out. Among 656 colonies, two colonies showed UGPase activities and three colonies for UNGPase activities. DNA sequence analyses and enzyme assays showed that two mutant clones (H145G) specifically have an UGPase activity, indicating that the changed glycine residue from histidine has the base specificity for UTP. Also, three double mutants (H145G/A325V) showed a UNGPase, and A325 was associated with sugar binding, conferring the specificity for the sugar substrates and V325 of the mutant appears to be indirectly involved in the binding of the N-acetylamine group of N-acetylglucosmine-1-phosphate. The authors Hosung Sohn and Yong-Sam Kim equally contributed to the study.     

Regulation of UDP-glucose pyrophosphorylase isozyme UGP5 associated with cold-sweetening resistance in potatoes

The regulation of UDP-Glc pyrophosphorylase (UGPase) isozyme, UGP5, was investigated in potato tuber. The cDNA for UGP5 was cloned into the bacterial expression vector pET21d and recombinant (RC) enzyme was expressed in E. coli (BL21 star cells). The RC-UGP5 isozyme was purified to near homogeneity using salt precipitation, hydrophobic interaction, and anion-exchange column chromatography. Kinetic analysis revealed that in the synthesis direction, K(m) values for Glc-1-P (0.83mM) and UTP (0.22mM) were similar to those observed previously with the mother tuber (MT)-UGP5. In the pyrophosphorolysis direction, the K(m) values for UDP-Glc (0.68mM) and PPi (0.56mM) were slightly higher than those observed previously. Maximum reaction velocities (V(max)) for RC-UGP5 were also elevated. Since the molecular mass, charge, and amino acid sequence of the MT- and RC-UGP5 isozymes were identical, it was assumed that altered kinetic constants may be due to an improper folding of RC-UGP5 polypeptide. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and proteomic analysis demonstrated that the UGP5 isozyme was a single polypeptide with a calculated molecular mass of 51.8kDa consisting of 477 amino acids. Native PAGE and kinetic analysis revealed that this polypeptide was monomeric in nature. Immunoblotting with specific antibodies and LC-MS/MS data indicated that UGP5 did not require any post-translational modification (e.g., phosphorylation, O-glycosylation, oligomerization/de-oligomerization, or the presence of the regulatory 14-3-3 proteins) for its regulation. Additionally, the two closely associated isozymes UGP5 and UGP6 in the cv. Snowden are likely the result of allelic differences of UGPase at a single locus.     

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 K_m with PPi (up to 33 mM), whereas Ncut deletions had greatly increased K_m 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 K_m 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.