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.     

Factors affecting oligomerization status of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6-9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.     

Control of Colanic Acid Synthesis

The nucleotide pools of certain mucoid, colanic acid-synthesizing strains of Escherichia coli, Salmonella typhimurium, and Aerobacter cloacae were examined, and in all cases the nucleotide sugars uridine-5'-diphosphate glucose (UDPG), uridine-5'-diphosphate galactose (UDPGal), guanosine-5'-diphosphate fucose (GDPF), and uridine-5'-diphosphate glucuronic acid (UDPGA) were detected. It is postulated that these nucleotide sugars are precursors in the synthesis of colanic acid. The levels of these nucleotide sugars and of the enzymes involved in their synthesis were examined in a number of mucoid strains and compared with the levels found in certain strains which were repressed in the synthesis of colanic acid, only becoming mucoid when grown in the presence of p-fluorophenylalanine (PFA). The levels of UDPG and UDPGal and the enzymes involved in their synthesis were substantially the same in both mucoid and repressed types, but the levels of UDPGA and GDPF and of some of the enzymes involved in their synthesis were much higher in mucoid strains. When repressed strains were grown in the presence of PFA, the levels of UDPGA and GDPF approached those found in mucoid strains. The existence of an operon, containing genes coding for certain key enzymes involved in colanic acid synthesis has been suggested.     

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。     

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...     

Substrate specificity and domain analyses of zeatin O-glycosyltransferases

The substrate specificity of two recombinant enzymes, zeatin O-glucosyltransferase 1 (ZOG1) and zeatin O-xylosyltransferase 1 (ZOX1), was further characterised. ZOG1 utilises zeatin (Z), UDPG, and UDPX as substrates to form O-glucosylzeatin (OGZ) and O-xylosylzeatin (OXZ) but has higher affinity to UDPG than UDPX. ZOX1 uses only UDPX, converting Z to OXZ. Dihydrozeatin (DHZ) is also a substrate for both enzymes, but only in combination with UDPX, giving rise to O-xylosyldihydrozeatin (OXDHZ). O-Glucosyldihydrozeatin (OGDHZ) is not formed by ZOG1, possibly due to steric hindrance. Regions relevant to UDPG/UDPX affinity and competition were identified using hybrid enzymes derived from domain exchanges of parental genes. The N-terminal half of the enzyme is important in this respect. The BstEII-BstAPI segment of ZOG1 correlates with inhibition of O-xylosyltransferase activity by UDPG while the BstAPI-Eco0109 segment of ZOG1 is required for utilisation of UDPG as the sugar donor.     

Enzymatic synthesis and reactions of uridine 5'-(5-thio-alphaD-glucopyranosyl pyrophosphate).

Uridine 5′-(5-thio-α-d-glucopyranosyl pyrophosphate), UDPTG, is an efficient substrate for yeast uridine 5′-(d-glucopyranosyl pyrophosphate), UDPG, pyrophosphorylase. K_m for UDPTG with the pyrophosphorylase is 0.2 mm and the analog reacts with a maximal velocity 96% that of UDPG. UDPTG is also a substrate for yeast UDP-galactose 4-epimerase. Although not a substrate for bovine liver UDPG dehydrogenase, UDPTG is a potent, mixed-type inhibitor with respect to both UDPG and nicotinamide adenine dinucleotide (NAD). UDPTG is synthesized in 30% yield from 5-thio-d-glucopyranose and in 85% yield from 5-thio-α-d-glucopyranose 1-phosphate by using mixtures of commercially available enzymes. The pK_a of the uracil moiety in UDPTG is the same as that in UDPG, and UDPTG appears to be similar to UDPG in the extent of secondary structural order. UDPTG, however, is more highly acid-labile than UDPG.     

Physicochemical and kinetic properties of unique isozymes of UDP-Glc pyrophosphorylase that are associated with resistance to sweetening in cold-stored potato tubers

Isozymes of UGPase with unique catalytic properties were purified from the cold-induced-sweetening (CIS) resistant cultivar Snowden (Solanum tuberosum). Two distinct peaks of UGPase activity were obtained when protein extracts were subjected to anion-exchange chromatography on DEAE-Sephacel. Polypeptides in the first eluted fraction (A-I) were ionically similar to the UGPase isozyme UGP3 previously purified and characterized from the cold-sweetening sensitive cultivar Norchip (Sowokinos et al. 1993, Plant Physiol 101: 1073-1080). Seventy-two percent of the total endogenous UGPase activity in Snowden (cv.) tubers, however, was found in a more basic protein fraction (A-II) that is not found in the Norchip cultivar. This study reports on the physicochemical and kinetic properties of these new polypeptides that demonstrate UGPase activity. The reaction in the direction of UDP-Glc synthesis was specific for the substrates Glc-1-P and UTP and there was an absolute requirement for Mg2+ ions. The catalytic properties of UGP5 were markedly different from UGPase isozymes previously described in terms of (1) affinity for the substrate Glc-1-P, (2) pH optimum, (3) maximum reaction velocity and (4) sensitivity to product inhibition with UDP-Glc. Chi-square analysis of fifty-four genetically diverse potato lines revealed that resistance to CIS was highly correlated with the presence of the A-II isozymes of UGPase. The kinetic properties of these unique forms of UGPase may underlie, in part, a tuber's ability to resist sweetening in the cold.     

Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and −0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.     

Zum Stoffwechsel der Stärke

Summary The determination of the activities of the enzymes which could be involved in the starch-sucrose conversion showed that the first step of this transfer is a phosphorolysis of starch. Both phosphorylases are able to attack the starch granules. The glu-l-P thus formed can be further metabolized to UDPG and fru-6-P, which are the substrates for the reaction catalysed by the sucrose-6-phosphate-synthetase. The activity of this enzyme increases about 4-fold during the period from immaturity to the first day of germination. The synthesis of sucrose-6-phosphate is inhibited by sucrose. This inhibition (causing an UDPG accumulation) provides a control for further starch breakdown.     

Cloning of cDNA for UDP-glucose pyrophosphorylase and the expression of mRNA in rice endosperm

Rice endosperm UDP-glucose pyrophosphorylase (UGPase) cDNA clones were isolated by screening a lambda ZAP II library prepared from poly (A(+)) RNA of japonica rice (cv Sasanishiki) endosperm with a probe of potato UGPase cDNA. One cDNA clone, possessing about 1,700 nucleotides, contained the complete open reading frame of rice UGPase. At the nucleotide-sequence level, the UGPase cDNA of rice endosperm had high homology with the UGPase cDNA of barley endosperm (84%) and potato tuber (71%). The calculated molecular weight (50 kDa) agrees with the value determined by SDS-PAGE (51 kDa). At the amino-acid sequence level, rice UGPase has high homology with the UGPase of barley (92%) and potato (85%). The enzyme contained conserved sequence elements which are thought to be involved in substrate binding and catalytic activity. A Southern-blot analysis indicated that the gene existed as a single copy. Expression of the enzyme in rice endosperm examined by Northern-blot analysis was high at 10-15 days after heading.     

U4 small nuclear RNA genes of trypanosomes: cloning of the Leptomonas seymouri gene and mutational analysis of core snRNP assembly

The enzyme UTP–glucose-1-phosphate uridylyltransferase (UDP–glucose pyrophosphorylase, UDPG:PP) is synthesized by practically all organisms, although prokaryotic UDPG:PPs are evolutionarily unrelated to the eukaryotic counterparts. The primary structure of prokaryotic UDPG:PPs is well conserved, although little information exists on the polymorphism of the genes coding for these enzymes. It has been reported that the galU gene encoding the Streptococcus pneumoniae UDPG:PP is absolutely required for the synthesis of the capsular polysaccharide, a sine qua non prerequisite for virulence. A 594 bp fragment covering 66% of the galU gene from 37 pneumococcal isolates and the type strains of Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus sanguinis, Streptococcus salivarius, and Streptococcus sobrinus has been amplified by PCR and sequenced. Up to 21 different alleles were found in S. pneumoniae. They possess a mosaic-like structure and belong to, at least, two evolutionarily distinct families that show a sequence divergence of 15–20%. In spite of its marked polymorphism, phylogenetic relationships among pneumococcal strains deduced from the galU gene matched those previously established by using alternative approaches. Comparison of the pneumococcal galU alleles with those from other streptococci indicated the existence of a complex network of genetic interchange. The galU gene represents an informative marker to be used alone or in conjunction with other molecular typing methods.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

Pyrophosphorylases in Solanum tuberosum (IV. Purification,Tissue Localization,and Physicochemical Properties of UDP-Glucose Pyrophosphorylase)

The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L. cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein. The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration. Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers. Leaves and roots contained lower levels of UGPase activity and protein. Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively. However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis. At physiological substrate concentrations (i.e. from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained. When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP). These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A. Conway, D.E. Koshland, Jr. [1968] Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP. The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T. Nakae [1971] J Biol Chem 246: 4404-4411). The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed.     

Open and closed structures of the UDP-glucose pyrophosphorylase from Leishmania major

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel beta-helix (LbetaH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.     

Cellulose synthesis by Acetobacter xylinum III. Matrix,primer and lipid requirements and heat stability of the cellulose-forming enzymes

The addition of soluble cellodextrins of increasing size to a cell envelope preparation of Acetobacter xylinum stimulated cellulose synthesis from UDPG. This stimulation was attributed to both acceptor and activator effects. Enzymes required for cellulose synthesis were found to be heat-unstable and those required for synthesis of glycosylated lipid components from UDPG, heat-stable. Both heat-inactivated envelope fragments and supernatant fluid from whole cells were necessary for cellulose synthesis from UDPG. Cellulose was not formed from UDPG in the presence of either supernatant fluid alone or heat-inactivated envelopes alone.The combined results of this and previous studies suggest that either the cell envelope is necessary for synthesis of a more immediate precursor to cellulose than UDPG, or that the synthesis from UDPG requires a matrix. The former suggestion and its possible link with lipid intermediate involvement was strengthened by the observation of inefficient glycoxylated lipid formation by a celluloseless mutant strain of A. xylinum. The possible locations of various enzyme activities required for the synthesis of the cellulose precursor are indicated and a possible microfibril nucleation process is discussed.     

Changes of Starch Synthetase Activity of Cucumber Leaves during Ammonium Toxicity

The changes of granule bound starch synthetase activity in cucumber leaves (Cucumis sativus L. cv. Suisei No. 2) were investigated during ammonium toxicity. Generally speaking the quantity of starch granules of injured plants were less than that of normal plants. ADPG is a more effective glucose donor to starch synthesis than UDPG. It was found that the starch synthetase activity of injured plants was decreased compared to the normal plants. This variation of enzyme activity was higher when UDPG was used as glucose donor. The addition of K⁺ and NH₄⁺ generally inhibited the enzyme activity when UDPG was used as glucose donor, but stimulated it when ADPG was used. This stimulation was found to be more effective in enzymes prepared from injured plants than from normal plants. The level of potassium bound to starch granules was not changed markedly between normal and injured plants.     

过量表达OsUgp2基因提高紫芝多糖含量

尿苷二磷酸葡萄糖焦磷酸化酶（UDP-glucose pyrophosphorylase,UGPase）是多糖生物合成过程中重要的酶,水稻基因组中存在两个UGPase同源基因分别命名为OsUgp1和OsUgp2。构建了由构巢曲霉3-磷酸甘油醛脱氢酶基因启动子驱动OsUgp2表达的真菌过量表达载体,并通过农杆菌介导法将OsUgp2基因转入紫芝中,获得了潮霉素抗性的转化菌株。PCR和Southern杂交结果显示OsUgp2基因成功整合到受体紫芝基因组中。半定量RT-PCR检测结果显示外源基因OsUgp2在紫芝转     

匍枝根霉纤维二糖合成酶胞内糖基供体的初探及结构功能研究

纤维二糖可有效诱导丝状真菌产纤维素酶,前期研究表明匍枝根霉Rhizopus stolonifer TP-02具有纤维二糖合成酶(CBS),可以尿苷二磷酸葡萄糖(UDPG)为糖基供体合成纤维二糖,从而开启纤维素酶的自诱导合成途径。为研究R. stolonifer中纤维二糖的胞内合成途径,通过重叠PCR在GDP-葡糖焦磷酸化酶基因ggp中引入硫胺吡啶抗性基因ptrA,分别转化原菌TP-02和△ugp突变株,构建△ggp和△ugp/△ggp突变株。利用液质联用(LC-MS)检测突变株的胞内糖组分,发现ggp的缺失对胞内纤维二糖合成的影响较弱,但同时缺失ugp则将直接导致二糖合成受阻。RT-qPCR结果显示△ggp突变株中纤维素酶基因转录水平较原株TP-02下调20%左右,而△ugp/△ggp突变株中被测基因的转录水平则出现了高达80%左右的下调。同时对突变株纤维素酶表达水平进行研究,发现△ugp/△ggp突变株中几乎检测不到纤维素酶活力。结果显示,UDPG为R. stolonifer胞内合成纤维二糖的主要糖基供体,而GDPG可能是UDPG的替代物,在UDPG不足时维持胞内二糖合成。此外,利用生物信息学方法对CBS结构功能深入分析,经丙氨酸扫描确定其合成纤维二糖的关键作用残基为Asp210和Asp300,为后续进一步研究及理性改造提供方向和理论依据。