Depression of guanosine diphosphate-mannose pyrophosphorylase by mutations in two different regulator genes involved in capsular polysaccharide synthesis in Escherichia coli K-12

Mutations in a regulator gene (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepress synthesis of guanosine diphosphate (GDP)-mannose pyrophosphorylase. In addition, a second mucoid mutation (capS, which maps separately from capR) also results in the derepression of GDP-mannose pyrophosphorylase. New conditions for assaying GDP-mannose hydrolyase and GDP-l-fucose synthetase permitted us to show that these enzymes are also derepressed in the capS mucoid strain. Although phosphomannose isomerase and uridine diphosphate-galactose-4-epimerase are derepressed in capR mucoid strains, they are not derepressed in capS mucoid strains. A nonmucoid mutant of a strain containing the capR9 (mucoid) allele was deficient in GDP-mannose pyrophosphorylase.     

Derepression of Phosphomannose Isomerase by Regulator Gene Mutations Involved in Capsular Polysaccharide Synthesis in Escherichia coli K-12

A regulator gene mutation (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepresses phosphomannose isomerase (PMI) synthesis. In contrast, a second mutation (capS, which maps separately from capR) that causes increased production of the same polysaccharide does not lead to increased synthesis of PMI (nor of several of the other enzymes involved in polysaccharide synthesis). Introduction of the capR9 allele by transduction or mutation of capR(+) to capR can change the phenotype of a mannose-negative nonmucoid strain to a mannose-positive mucoid phenotype. Thus, genotype capR(+)man-2 is mannose-negative and nonmucoid, but genotype capR9 man-2 is mannose positive and mucoid. Other interactions between these alleles in the synthesis of capsular polysaccharide are recorded.     

Dominance of Ultraviolet Radiation Resistance in Partial Diploids of Escherichia coli K-12

Although an F'13 capR(+)/capR9 strain is nonmucoid and an F'13 capR9/capR(+) strain is mucoid, both strains are ultraviolet (UV)-resistant. In contrast, haploid capR9 strains are UV-sensitive. Therefore, UV resistance is dominant to UV sensitivity, regardless of whether the capR(+) allele is on the chromosome or on the F'13 episome.     

Pl-mediated Transduction of a Gene That Controls Radiation Sensitivity and Capsular Polysaccharide Synthesis from Shigella dysenteriae to Escherichia coli

When Shigella dysenteriae strain 60 is used as a donor and Escherichia coli K-12 strains that are ultraviolet (UV)-sensitive, mucoid, and proline-requiring (Pro(-)) are employed as recipients, selection for Pro(+) yields 2 to 6% nonmucoid clones. All of the nonmucoid clones examined are UV-resistant. Most of the nonmucoid UV-resistant transductants are partial diploids for the genes being studied. When these Shigella-Escherichia hybrids are used as donors with the same E. coli recipients, the cotransduction of Pro(+) and nonmucoidness is greatly increased (59 to 94% cotransduction). All of these nonmucoid transductants examined were also UV-resistant. The results indicate that Shigella contains an allele (designated ShproC(+)) homologous to proC of E. coli and a second linked allele (designated ShcapR(+)) homologous to the capR allele of E. coli. The ShcapR(+) allele changes the phenotype of certain E. coli strains from mucoid UV-sensitive (capR6) or very sensitive (capR9) to nonmucoid and UV-resistant. Unanticipated capR allele interactions in the partial diploid hybrids are described.     

Uridine diphosphate glucose dehydrogenase from cornea and epiphysial-plate cartilage

1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the K(m) values for UDP-glucose and NAD(+) were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.     

基于双酶偶联法合成尿苷二磷酸葡萄糖醛酸的研究

尿苷二磷酸（uridine diphosphate,UDP）-葡萄糖醛酸是细胞内重要的糖基供体,参与多种代谢途径,也是体外进行糖基化反应的重要糖基供体,但其价格昂贵、工艺复杂,限制了其大量使用,无法满足生产需求。基于此,利用双酶偶联法氧化UDP-葡萄糖生成UDP-葡萄糖醛酸,并研究反应产物的合成情况。以UDP-葡萄糖为底物、烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD+）为辅因子,利用化脓性链球菌Streptococcus pyogenes源的尿苷二磷酸葡萄糖脱氢酶（UDP-glucose dehydrogenase,UGD）、猪源的乳酸脱氢酶（lactate dehydrogenase,LDH）,双酶偶联催化合成UDP-葡萄糖醛酸,并通过高效液相色谱、质谱及核磁共振氢谱对反应产物进行检测,确定产物的结构及产物的生成量。结果表明,利用双酶偶联法氧化UDP-葡萄糖所得到的产物为UDP-葡萄糖醛酸。在UGD的作用下,氧化UDP-葡萄糖生成UDP-葡萄糖醛酸,同时辅因子NAD+在LDH的作用下实现循环再生,减少高能产物辅酶还原型烟酰胺腺嘌呤二核苷酸（reduced nicotinamide adenine dinucleotide,NADH）对反应的反馈抑制作用,产物的生成率约为60.17%。研究提高了产物UDP-葡萄糖醛酸产物生成量,为后续工业化制备提供了新思路。     

Multiple regulator gene control of the galactose operon in Escherichia coli K-12

Previous studies showed that nonsense mutations in either of two genes (capR or capS) or an undefined mutation in a third gene (capT) led to pleiotropic effects: (i) increased capsular polysaccharide synthesis (mucoid phenotype); (ii) increased synthesis of enzymes specified by at least four spatially separated operons involved in synthesis of capsular polysaccharide including the product of the galE gene, UDP-galactose-4-epimerase (EC 5.1.3.2) in capR mutants. The present study demonstrated that the entire galactose (gal) operon (galE, galT, and galK) is derepressed by mutations in either the capR or the capT genes, but not by mutation in capS. Double mutants (capR9 capT) were no more derepressed than the capR9 mutant, indicating that capR9 and capT regulate the gal operon via a common pathway. Isogenic double mutants containing either galR(+), galR(-), galR(s), or galO(c) in combination with either capR(+) or capR9 were prepared and analyzed for enzymes of the gal operon. The results demonstrated that capR9 caused derepression as compared to capR(+) in all of the combinations. Strains with a galR(s) mutation are not induced, for the gal operon, by any galactose compound including d-fucose, and this was confirmed in the present study using d-fucose. Nevertheless, the derepression of galR(s) capR9 compared to galR(s) capR(+) was four- to sixfold. The same derepression was observed when galR(+)capR9 was compared to galR(+)capR(+). The data eliminate the explanation that internal induction of the gal operon by a galactose derivative was causing increased gal operon enzyme synthesis in capR or capT mutants. Furthermore, the same data suggest that the galR and capR genes are acting independently to derepress the gal operon. A modified model for the structure of the gal operon is proposed to explain these results. The new feature of the model is that two operator sites are suggested, one to combine with the galR repressor and one to combine with the capR repressor.     

Uridine diphosphate glucuronic acid production and utilization in various tissues actively synthesizing glycosaminoglycans

1. UDP-glucose dehydrogenase has been partially purified from sheep nasal septum cartilage, neonatal rat skin and bovine corneal epithelium. 2. The pH profile, K(m) values for NAD(+) and UDP-glucose, activation energy and molecular weight have been determined for the enzyme from several of the tissues. 3. The sugar nucleotide concentrations in each of the tissues have been related to the spectrum of glycosaminoglycans produced by each tissue. 4. The presence of an allosteric UDP-xylose-binding site distinct from the active site(s) in sheep nasal septum UDP-glucose dehydrogenase has been demonstrated. 5. An active UDP-glucuronic acid nucleotidase has been demonstrated in sheep nasal cartilage. 6. Tissue-space experiments have shown the cell water content of sheep nasal septum cartilage to be 14% of the wet weight. 7. Glucuronic acid 1-phosphate does not occur in measurable amounts in sheep nasal septum cartilage and no UDP-glucuronic acid pyrophosphorylase activity could be detected in this tissue. 8. The inhibition by UDP-xylose with respect to both substrates, UDP-glucose and NAD(+), has been examined, and shown to be allosteric.     

Characterization of UDP-glucose dehydrogenase isoforms in the medicinal legume Glycyrrhiza uralensis

Uridine 5′-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1–4) showed UGD activity in vitro. GuUGD1–4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1–4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.     

Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.     

Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls

Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD(+) ( approximately 40 microM) but variable affinities for the substrate UDP-glucose (120-335 microM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes.     

Uridine diphosphoglucose pyrophosphorylase activity and differentiation in the cellular slime mold Physarum polycephaluno

The specific activity of uridine 5'-triphosphate:alpha-d-glucose 1-phosphate uridyltransferase (EC 2.7.7.9) (also called uridine 5'-diphosphate [UDP]-glucose pyrophosphorylase) has been found to increase up to eightfold during spherule formation by the slime mold Physarum polycephalum. The enzyme accumulates during the first 8 to 9 h after initiation of spherule formation, declines to basal levels found in vegetative microplasmodia by 15 h, and is undetectable in completed spherules. Specific activities for UDP-glucose pyrophosphorylase in vegetative microplasmodia range from 15 to 30 nmol of UDP-glucose formed per min per mg of protein, whereas accumulated levels during spherule formation can attain a specific activity as high as 125 nmol of UDP-glucose formed per min per mg of protein. The scheduling and extent of accumulation are critically dependent on an early log-phase age of microplasmodia originally induced to form spherules. Spherule induction by 0.2 M or 0.5 M mannitol delays this schedule in a variable and unpredictable manner. Spherule-forming microplasmodia which have accumulated high levels of UDP-glucose pyrophosphorylase spontaneously excrete the enzyme when transferred to salts medium containing 0.2 M or 0.5 M mannitol. The excreted enzyme is subsequently destroyed or inactivated. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of UDP-glucose pyrophosphorylase requires concomitant protein synthesis and prior ribonucleic acid synthesis.     

Changes in enzyme activities involved in formation and interconversion of UDP-sugars during the cell cycle in a synchronous culture of Catharanthus roseus

Changes in the activities of enzymes involved in UDP-sugar formation [UDP-glucose pyrophosphorylase (EC 2.7.7.9), sucrose synthase (EC 2.4.1.13) and UDP-glucuronic acid pyrophosphorylase (EC 2.7.7.44)], and interconversion [UDP-glucuse 4-epimerase (EC 5.1.3.2), UDP-glucose dehydrogenase (EC 1.1.1.22), UDP-glucuronic acid decarboxylase (EC 4.1.1.35) and UDP-xylose 4-epimerase (EC 5.1.3.5)] were investigated during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. The specific activities of UDP-glucose pyrophosphorylase and UDP-glucose 4-epimerase increased in the G2 phase before the first cell division, and those of sucrose synthase, UDP-glucose dehydrogenase and UDP-glucuronic acid pyrophosphorylase increased in the G1 phase after the first cell division. However, during the cell cycle, UDP-glucuronic acid decarboxylase and UDP-xylose 4-epimerase did not change significantly in their specific activities. Changes in enzyme activities are discussed in relation to those reported previously for cell wall composition (S. Amino et al. 1984. Physiologia Plantarum 60: 326–332).     

A microassay for UDP-glucose dehydrogenase

An assay for UDP-glucuronic acid [J. Singh, L. R. Schwarz, and F. J. Wiebel, Biochem. J. 189, 369–372 (1980)] has been utilized for determining UDP-glucose dehydrogenase activity. The assay for UDP-glucuronic acid, a product of UDP-glucose dehydrogenase, is based on the fluorometric determination of -glucuronosyl benzo(a)pyrene. This compound is formed from UDP-glucuronic acid and 3-hydroxybenzo(a)pyrene in a reaction catalyzed by the glycuronosyl transferase of guinea pig microsomes. Unreacted 3-hydroxybenzo(a)pyrene is removed by extraction with chloroform-methanol, and the amount of gluconosylbenzo(a)pyrene formed is determined fluorometrically. Because this assay for UDP-glucose dehydrogenase is about 500 times more sensitive than spectrophotometric assays, it can be used to measure the amount of enzyme extractable from milligram quantities of connective tissue. Some kinetic properties of UDP-glucose dehydrogenase extracted from rabbit tissue have been determined. No evidence of different forms of the enzyme in rabbit liver, cartilage, or corneal stroma was found.     

Uridine diphosphate-sugar metabolism by sycamore (Acer pseudoplatanus) cambial tissue

Summary Extracts of sycamore cambial tissue convert UDP-glucose into UDP-glucuronic acid, and the latter into UDP-xylose and UDP-rhamnose. None of the corresponding galactose series of monosaccharides was formed indicating the absence of epimerases, postulated as an important feature of differentiation. UDP-glucose is formed in greater quantities than any of its nucleoside analogues and it is suggested that UDP-glucose plays a more important role in carbohydrate metabolism in this tissue.     

Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.     

Active site residues and mechanism of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase catalyzes the NAD+-dependent twofold oxidation of UDP-glucose to give UDP-glucuronic acid. A sequestered aldehyde intermediate is produced in the first oxidation step and a covalently bound thioester is produced in the second oxidation step. This work demonstrates that the Streptococcus pyogenes enzyme incorporates a single solvent-derived oxygen atom during catalysis and probably does not generate an imine intermediate. The reaction of UDP-[6",6"-di-2H]-d-glucose is not accompanied by a primary kinetic isotope effect, indicating that hydride transfer is not rate determining in this reaction. Studies with a mutant of the key active site nucleophile, Cys260Ala, show that it is capable of both reducing the aldehyde intermediate, and oxidizing the hydrated form of the aldehyde intermediate but is incapable of oxidizing UDP-glucose to UDP-glucuronic acid. In the latter case, a ternary Cys260Ala/aldehyde intermediate/NADH complex is presumably formed, but it does not proceed to product as both release and hydration of the bound aldehyde occur slowly. A washout experiment demonstrates that the NADH in this ternary complex is not exchangeable with external NADH, indicating that dissociation only occurs after the addition of a nucleophile to the aldehyde carbonyl. Studies on Thr118Ala show that the value of kcat is reduced 160-fold by this mutation, and that the reaction of UDP-D-[6",6"-di-2H]-glucose is now accompanied by a primary kinetic isotope effect. This indicates that the barriers for the hydride transfer steps have been selectively increased and supports a mechanism in which an ordered water molecule (H-bonded to Thr118) serves as the catalytic base in these steps.     

Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene.

The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide precursors. The introduction and overexpression of the cloned P. aeruginosa phosphomannose isomerase (pmi) gene in both mucoid and nonmucoid strains led not only to the appearance of PMI levels in cell extracts several times higher than those present in the wild-type mucoid strain, but also in higher PMM and GMP specific activities. In extracts of both strains, however, the specific activity of GMD did not change as a result of pmi overexpression. In contrast, the introduction of the cloned Escherichia coli manA (pmi) gene in P. aeruginosa caused an increase in only PMI and PMM activities, having no effect on the level of GMP. This suggests that an increase in PMI activity alone does not induce high GMP activity in P. aeruginosa. The heterologous overexpression of the P. aeruginosa pmi gene in the E. coli manA mutant CD1 led to the appearance in cell extracts of not only PMI activity but also GMP activity, both of which are normally undetectable in extracts of CD1. We discuss the implications of these results and propose a mechanism by which overexpression of the P. aeruginosa pmi gene can cause an elevation in both PMM and GMP activities.