Role In Sugar Metabolism Research Articles

Crystal structure of the S. cerevisiae D-ribose-5-phosphate isomerase: comparison with the archaeal and bacterial enzymes

Ribose-5-phosphate isomerase A has an important role in sugar metabolism by interconverting ribose-5-phosphate and ribulose-5-phosphate. This enzyme is ubiquitous and highly conserved among the three kingdoms of life. We have solved the 2.1 Å resolution crystal structure of the Saccharomyces cerevisiae enzyme by molecular replacement. This protein adopts the same fold as its archaeal and bacterial orthologs with two α/β domains tightly packed together. Mapping of conserved residues at the surface of the protein reveals strong invariability of the active site pocket, suggesting a common ligand binding mode and a similar catalytic mechanism. The yeast enzyme associates as a homotetramer similarly to the archaeal protein. The effect of an inactivating mutation (Arg189 to Lys) is discussed in view of the information brought by this structure.

Isolation and functional characterization of a novel plastidic hexokinase from Nicotiana tabacum

Due to their central role in sugar metabolism and signalling plant hexokinases have been studied in great detail, however, little is known about the spatial and temporal expression and the sub-cellular distribution of individual hexokinase isoforms. Based on in planta and in vitro studies the recently isolated tobacco hexokinase 2 (Hxk2) could be located in the chloroplast stroma and biochemically characterized. Hxk2 represents the first innerplastidic hexokinase described from higher plants. Promoter studies indicate that Hxk2 is mainly expressed in cells of the vascular starch sheath and xylem parenchyma, in guard cells and root tips. We propose a role for Hxk2 in starch and secondary metabolism in the mentioned tissues.

Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli.

UDP-galactose 4-epimerase plays a critical role in sugar metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose. Originally, it was assumed that the enzyme contained a "traditional" catalytic base that served to abstract a proton from the 4'-hydroxyl group of the UDP-glucose or UDP-galactose substrates during the course of the reaction. However, recent high-resolution X-ray crystallographic analyses of the protein from Escherichia coli have demonstrated the lack of an aspartate, a glutamate, or a histidine residue properly oriented within the active site cleft for serving such a functional role. Rather, the X-ray crystallographic investigation of the epimerase.NADH.UDP-glucose abortive complex from this laboratory has shown that both Ser 124 and Tyr 149 are located within hydrogen bonding distance to the 4'- and 3'-hydroxyl groups of the sugar, respectively. To test the structural role of Ser 124 in the reaction mechanism of epimerase, three site-directed mutant proteins, namely S124A, S124T, and S124V, were constructed and crystals of the S124A.NADH.UDP, S124A.NADH.UDP-glucose, S124T. NADH.UDP-glucose, and S124V.NADH.UDP-glucose complexes were grown. All of the crystals employed in this investigation belonged to the space group P3221 with the following unit cell dimensions: a = b = 83.8 A, c = 108.4 A, and one subunit per asymmetric unit. X-ray data sets were collected to at least 2.15 A resolution, and each protein model was subsequently refined to an R value of lower than 19.0% for all measured X-ray data. The investigations described here demonstrate that the decreases in enzymatic activities observed for these mutant proteins are due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural perturbations. In addition, these structures demonstrate the importance of a hydroxyl group at position 124 in stabilizing the anti conformation of the nicotinamide ring as observed in the previous structural analysis of the epimerase.NADH. UDP complex.

Water-stress response in aspen ( Populus tremula): Differential accumulation of dehydrin, sucrose synthase, GAPDH homologues, and soluble sugars

Summary In a previous study we identified a novel 66-kD boiling-stable protein (BspA) that accumulates in cultured shoots of aspen ( Populus tremula L.) in response to gradual water stress and ABA application, as well as to osmotic and cold stresses (Altman et al., 1996; Pelah et al., 1995). BspA was found to be the major heat-stable water stress-responsive protein in aspen. Here, the expression of other water-stress responsive proteins — DSP 16 (dehydrin), cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and sucrose synthase — was studied in aspen roots and shoots. These proteins were identified immunologically using polyclonal antibodies isolated from the resurrection plant Craterostigma plantagineum . A 43-kD homologue and a 31-kD homologue of dehydrin were found to accumulate in roots and shoots of aspen after water stress in a tissue specific manner. Shoots and roots of intact plantlets subjected to ABA application accumulated a 33-kD dehydrin homologue. A sucrose synthase homologue (90 kD) accumulated in water-stressed shoots and was constitutively present at high levels in control and water-stressed roots harvested from intact plandets. GAPDH, a key enzyme in the glycolysis and gluconeogenesis pathways, exhibited some accumulation in shoots in response to water stress. Sugar analysis revealed that water stress results in increased sucrose and decreased glucose levels in the aspen leaves. The data presented here suggest that the water-stress-response mechanisms present in herbaceous plants are also present in woody plants. Woody plants subjected to water stress accumulate dehydrin-like proteins and specific enzymes such as sucrose synthase and GAPDH, which may play a dominant role in sugar metabolism under these conditions.

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing α-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47

Thermoactinomyces vulgaris R-47 produces two α-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and α- and β-cyclodextrins. Whereas k cat/ K m values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely.A TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

Expression of mitochondrial genes and DNA-repair-related nuclear genes is altered in xeroderma pigmentosum fibroblasts.

Differential hybridization was used to detect repair defects in xeroderma pigmentosum (XP) that are not amenable to current analyses. cDNA libraries were constructed from cytoplasmic RNA of normal and XP fibroblast strains (complementation groups A and D) and analyzed for differential gene expression. More than 40,000 lambda gt10 cDNA clones were differentially screened with in vitro transcripts made from cDNA in the pBluescript vector. Six differential clones were detected in the libraries of the XP group A and D strains which caused stronger or weaker signals when probed with transcripts from XP strains than with those from the normal strains. Two clones coded for mitochondrial genes: mitochondrial 16 S rRNA and ATPase 6L. Overexpression of mitochondrial genes in XP may indicate that functions of the ATP-generating system are impaired since such functions are intensified whenever they become insufficient, for example as a consequence of DNA damage. It is tempting to assume that abnormal mitochondria are one of the causes for the neurological malfunctions in XP. Furthermore, densitometric analysis of Northern blots revealed that mRNA of lactate dehydrogenase, chain M, was less abundant in four XP group A strains (extent of reduction: 70%) and in two XP group D strains (extent of reduction: 58%). Enzyme activity was also diminished. In addition, mRNA of the gene for glyceraldehyde-3-phosphate dehydrogenase was less expressed in the same XP group A and D fibroblast strains investigated (reduction in both complementation groups: 50%). Both glycolytic enzymes have nuclear functions apart from their role in sugar metabolism. Lactate dehydrogenase, chain M, is identical to a helix-destabilizing protein; it is closely associated with chromatin and unfolded DNA, suggesting a role in DNA synthesis and transcription. The 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase is involved in transcription and was shown to be identical to uracil-DNA glycosylase, a base-excision repair enzyme. We presume that the nuclear functions of these glycolytic enzymes may be thwarted in the XP strains investigated and may account for malfunctions in XP, particularly for neurological disturbances.

Characterization of Tobacco Protein Kinase NPK5, a Homolog ofSaccharomyces cerevisiaeSNF1 That Constitutively Activates Expression of the Glucose-RepressibleSUC2Gene for a Secreted Invertase ofS. cerevisiae

We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells. The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells. SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source. Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S. cerevisiae. On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene. These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S. cerevisiae. The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions. Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family. NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants. The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.

Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae.

We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells. The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells. SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source. Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S. cerevisiae. On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene. These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S. cerevisiae. The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions. Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family. NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants. The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.

Pyrophosphate-dependent phosphofructokinase from pollen: properties and possible roles in sugar metabolism

Pyrophosphate-dependent phosphofructokinase from pollen: properties and possible roles in sugar metabolism

Pyrophosphate‐dependent phosphofructokinase from pollen: properties and possible roles in sugar metabolism

To evaluate the role of pyrophosphate‐dependent phosphofructokinase (PFP. EC 2.7.1.90) in the sugar metabolism of pollen. its occurrence and properties were studied in pollen grains of several plants including camellia (Camellia japonica L.). In all pollen samples, PFP was strongly activated by fructose‐2,6‐bisphosphate (F2,6BP), and the activity of F2,6BP‐activated PFP was higher than that of phosphofructokinase (PFK. EC 2.7.1.11). PFP partially purified from camellia pollen required Mg2+ for activity with an optimum at 1 mM. and was almost unaflected by a variety of metabolites at 1 mM. Its molecular mass was around 220 kDa, and apparent Km values for F6P, PPi. F1, 6BP and Pi were 294, 4, 20 and 580 uM, respectively. The levels of F2.6BP. PPi and F6P in camellia pollen were sufficent to support the forward reaction by PFP, and PFP, was 20‐ to 40‐fold more active than PFK during pollen growth. These results suggest that pollen PFP plays a role in glycolysis but not gluconeogenesis. and the possible relevance of this to pollen tube growth is discussed.

A hyperglycemic factor extracted from the pancreas.

THE metabolic processes of the body are governed by compensating checks and balances which are frequently interdependent, some accelerating, some inhibiting specific processes. Among the most extensively studied of these activities is that of the factors, affecting carbohydrate metabolism. Insulin has, of course, been long known to be of prime importance to this process, but investigations have suggested that another substance obtained from the pancreas may also play a role in sugar metabolism. This substance has been found to produce hyperglycemia and increase the rate of hepatic glycogenolysis. There is at present no direct proof that the pancreatic hyperglycemic factor is functional under physiologic conditions. Results of cross-circulation experiments by Weinstein, Smith and Foa (1) have suggested that this may be true, but further observations are deemed necessary. Certain other evidence, both clinical and experimental, strongly supports this possibility and shall be briefly discussed at this time.