Inhibition of phosphoglucose isomerase allozymes from the wing polymorphic waterstrider,Limnoporus canaliculatus,by pentose shunt metabolites

Inhibition of phosphoglucose isomerase (PGI) allozymes from the wing-polymorphic waterstrider, Limnoporus canaliculatus, by three pentose-shunt metabolites was studied at several different temperatures. This was done to determine if the allozymes exhibited a differential ability to participate in lipid biosynthesis via differential partitioning of carbon flux through the pentose shunt versus glycolysis. 6-Phosphogluconate and erythrose-4-phosphate proved to be strong competitive inhibitors of PGI, while sedoheptulose-7-phosphate was a very weak inhibitor. The PGI allozymes from L. canalicualtus were differentially inhibited by 6-phosphogluconate at two of the three temperatures studied. However, this property does not appear to be an adaptive difference between the allozymes but, rather, a correlated effect resulting from variation in substrate binding. Estimates of reaction rates for the allozymes indicate that the differences in inhibition result in no detectable differences in reaction velocities. Thus, no evidence in support of the hypothesis that PGI allozymes from Limnoporus canaliculatus were adapted to function in different metabolic capacities via differential inhibition was obtained in this study. However, the importance of this characteristic in allozymic adaptation in natural populations remains an open question.Supported by NSF Grant DEB 7908802 and UPHS Grant GM 21133 to R. K. Koehn and an NSF dissertation improvement grant to A. J. Zera.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase     

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum. Segments of young and mature leaves of pea released the carbons of glucose-[¹⁴C] as ¹⁴CO₂ in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1-¹⁴C], -[2-¹⁴C], -[3,4-¹⁴C], and -[6-¹⁴C] was determined and showed that the high yield of CO₂ from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.     

Light and dark assimilation of nitrate in plants

Abstract. Heterotrophic assimilation of nitrate in roots and leaves in darkness is closely linked with the oxidative pentose phosphate pathway. The supply of glucose-6-phosphate to roots and chloroplasts in leaves in darkness is essential for assimilation of nitrite into amino acids. When green leaves are exposed to light, the key enzyme, glucoses-phosphate dehydrogenase, is inhibited by reduction with thioredoxin. Hence the dark nitrate assimilatory pathway is inhibited under photoautotrophic conditions and replaced by regulatory reactions functioning in light. On account of direct photo-synthetic reduction of nitrite in chloroplasts and availability of excess NADH for nitrate reduclase, the rate of nitrate assimilation is extremely rapid in light. Under dark anaerobic conditions also nitrate is equally rapidly reduced to nitrite on account of abolition of competition for NADH between nitrate reductase and mitochondrial oxidation.     

No contribution of the pentose phosphate pathway in dormancy-breaking of cocklebur seeds

The role of the oxidative pentose phosphate (PP) pathway in the dormancy-breaking of cocklebur (Xanthium pennsylvanicum Wallr.) seeds was investigated. D-[1-¹⁴C]-glucose or D-[6-¹⁴C]-glucose was fed to dormant and non-dormant lower seeds or to their axial or cotyledonary segments which were imbibed for different durations, and C₆/C₁ ratios of respired ¹⁴CO₂ as an index of the PP pathway activity were calculated. Contrary to expectation, there was no significant difference in the C₆/C₁ ratios between the dormant and non-dormant seeds or segments during a water imbition period of 24 h, although the PP pathway actually operated already in an early stage of water imbibition. Also concerning the activities of G6PDH and 6PGDH, the key enzymes of this pathway, no difference between the dormant and non-dormant seeds was found. It was thus concluded that, unlike other seeds, there is no contribution of the PP pathway to the regulation of dormancy of the cocklebur seed.     

Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum

1. Cell-free extracts of the photosynthetic bacterium Chlorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase-the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO₂ fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate-the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.Support of a J. S. Guggenheim Fellowship is gratefully acknowledged     

The function of the pentose phosphate pathway in Phaseolus mungo hypocotyls

The metabolism of d-gluconate-[1-¹⁴C] and -[6-¹⁴C] by segments from etiolated hypocotyls of Phaseolus mungo has been studied. The release of ¹⁴CO₂ from gluconate-[1-¹⁴C] was greater than that from gluconate-[6-¹⁴C] in all parts of hypocotyls examined. Incorporation of the radioactivity from gluconate-[6-¹⁴C] into RNA, lignin and aromatic amino acid fractions was greater in the upper (younger) part of the hypocotyls. Incorporation into sugars was greater in the lower (more mature) parts.     

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L⁻¹ l-lysine from mannitol at a yield of 0.26 mol mol⁻¹ and a maximum productivity of 2.1 g L⁻¹ h⁻¹. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol⁻¹ using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol⁻¹. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.     

转酮酶在癌症中的作用及其机制研究进展

肿瘤细胞的一大重要特征是代谢水平的改变。戊糖磷酸途径作为细胞产生NADPH和五碳糖的主要通路,在肿瘤发生发展过程中发挥着重要功能。转酮酶是戊糖磷酸途径中的关键酶之一,越来越多的研究表明其与癌症病人预后具有显著相关性。作为肿瘤诊断、治疗的潜在靶标,转酮酶具有重要的研究价值。我们就目前癌症研究中对转酮酶的研究进展做简要综述。