Calvin cycle genes in Nitrobacter vulgaris T3

Abstract The genes encoding the Calvin cycle enzymes of Nitrobacter vulgaris T3 are found as two separate clusters on the chromosome. One cluster contains the genes for the large and small subunits of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), glyceraldehyde-3-phosphate dehydrogenase, and one encoding a regulatory protein of the LysR family. The other cluster contains the genes for fructose-1,6-/sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, and fructose-1,6-/sedoheptulose-1,7-biphosphate aldolase. With the exception of the LysR-like gene, the genes in each cluster are apparently transcribed in the same direction. The deduced amino acid sequence of both the large and small subunits of RuBisCO are most similar (84–86%) to those of Thiobacillus ferrooxidans and Chromatium vinosum . The deduced sequences of phosphoribulokinase and fructose/sedoheptulose bisphosphatase are 67–73 aand 44–46% similar to those reported for other autotrophic bacteria, respectively.     

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.     

Biochemical changes during germination and seedling growth in Cuscuta campestris

Changes in the contents of starch, protein, DNA, RNA, total phosphorus, acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis.
Starch content in Cuscuta seeds showed a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination and then decreased. Total phosphorus in the seeds remained almost unchanged during the period of study. Both trichloroacetic acid soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed.     

Comparative proteomes of Corynebacterium glutamicum grown on aromatic compounds revealed novel proteins involved in aromatic degradation and a clear link between aromatic catabolism and gluconeogenesis via fructose-1,6-bisphosphatase

The current study examined the aromatic degradation and central metabolism in Corynebacterium glutamicum by proteomic and molecular methods. Comparative analysis of proteomes from cells grown on gentisate and on glucose revealed that 30% of the proteins of which their abundance changed were involved in aromatic degradation and central carbon metabolism. Similar results were obtained from cells grown on benzoate, 4-cresol, phenol, and resorcinol. Results from these experiments revealed that (i) enzymes involved in degradation of benzoate, 4-cresol, gentisate, phenol, and resorcinol were specifically synthesized and (ii) that the abundance of enzymes involved in central carbon metabolism of glycolysis/gluconeogenesis, pentose phosphate pathway, and TCA cycles were significantly changed on various aromatic compounds. Significantly, three novel proteins, NCgl0524, NCgl0525, and NCgl0527, were identified on 4-cresol. The genes encoding NCgl0525 and NCgl0527 were confirmed to be necessary for assimilation of 4-cresol with C. glutamicum. The abundance of fructose-1,6-bisphosphatase (Fbp) was universally increased on all the tested aromatic compounds. This Fbp gene was disrupted and the mutant WT(Deltafbp) lost the ability to grow on aromatic compounds. Genetic complementation by the Fbp gene restored this ability. We concluded that gluconeogenesis is a necessary process for C. glutamicum growing on various aromatic compounds.     

Enzyme co-localization in pea leaf chloroplasts: glyceraldehyde-3-P dehydrogenase,triose-P isomerase,aldolase and sedoheptulose bisphosphatase

Nearest neighbor analysis of immunocytolocalization experiments indicates that the enzymes glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase are located close to one another in the pea leaf chloroplast stroma, and that aldolase is located close to sedoheptulose bisphosphatase. Direct transfer of the triose phosphates between glyceraldehyde-3-P dehydrogenase and triose-P isomerase, and from glyceraldehyde-3-P dehydrogenase and triose-P isomerase to aldolase, is then a possibility, as is direct transfer of sedoheptulose bisphosphate from aldolase to sedoheptulose bisphosphatase. Spatial organization of these enzymes may be important for efficient CO₂ fixation in photosynthetic organisms. In contrast, there is no indication that fructose bisphosphatase is co-localized with aldolase, and direct transfer of fructose bisphosphate from aldolase to fructose bisphosphatase seems unlikely.     

Mononuclear and polymorphonuclear leukocytes show increased fructose-1,6-bisphosphatase activity in patients with type 1 diabetes mellitus

The activity and localization of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in blood leukocytes of patients with type 1 diabetes mellitus and healthy adults were investigated immunocytochemically. The amount of polymorphonuclear (PMN) and mononuclear (MN) cells with positive FBPase immunocytochemical reaction was 57% and 68%, respectively, in pathological, and 38% and 42%, respectively, in healthy donors. Results of light microscopic investigations were confirmed by measurements of FBPase activity following lysis of PMN and MN cells. The enzyme activity of PMN and MN leukocytes was higher in diabetes mellitus than in healthy adults, by 30% and 127%, respectively. Using immunocytochemistry together with electron microscopy, FBPase was detected not only in the cytoplasm but also in the nucleus of leukocytes of both patients with insulin-dependent diabetes mellitus and healthy donors.     

Evolutionary History of the Enzymes Involved in the Calvin–Benson Cycle in Euglenids

Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin–Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin–Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin–Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast.     

Crystal structure of heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding

The heart‐specific isoform of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well‐known inhibitor of PFKFB2, co‐crystallized in the 2‐kinase domains of both orthologues, occupying the fructose‐6‐phosphate binding‐site and extending into the γ‐phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ‐phosphate site by citrate proved highly consequential to the binding of co‐complexed ATP analogues. The bovine structure, which co‐crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co‐complexed with AMPPNP, which, unlike ADP, contains a γ‐phosphate. The presence of this γ‐phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding‐pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed‐back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117–124. © 2016 Wiley Periodicals, Inc.     

Photosynthetic electron transport and carbon-reduction-cycle enzyme activities under long-term drought stress in Casuarina equisetifolia Forst. & Forst.

The inhibition of photosynthetic electron transport and the activity of photosynthetic carbon reduction cycle (PCR) enzymes under long-term water stress after slow dehydration was studied in non-nodulated Casuarina equisetifolia Forst. & Forst. plants. Initially, drought increased the fraction of closed Photosystem II (PS II) reaction centres (lowered qP) and decreased the quantum yield of PS II electron transport (_PSII) with no enhancement of non-radiative dissipation of light energy (qN) because it increased the efficiency of electron capture by open PS II centres (F_v/F_m). As drought progressed, F_v/F_m fell and the decrease in _PSII was associated with an increased qN. The kinetics of dark relaxation of fluorescence quenching pointed to an increase in a slowly-relaxing component under drought, in association with increased contents of zeaxanthin and antheraxanthin. Total NADP-dependent malate dehydrogenase activity increased and total stromal fructose-1,6-bisphosphatase activity decreased under drought, while the activation state of these enzymes remained unchanged. Water stress did not alter the activity and the activation state of ribulose bisphosphate carboxylase oxygenase.