Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides

In Rhodobacter sphaeroides, many of the structural genes encoding enzymes of the Calvin cycle are duplicated and grouped within two separate clusters. In this study, the nucleotide sequence of a 5627-base pair region of DNA that contains the form I Calvin cycle gene cluster has been determined. The five open reading frames are arranged in the order, fbpA prkA cfxA rbcL rbcS and are tightly linked and oriented in the same direction. The results of insertional mutagenesis studies suggest the genes are organized within an operon. Consistent with this proposal, the cfxA gene has been tentatively identified as a gene encoding the Calvin cycle enzyme, aldolase. Measurement of the activities of various Calvin cycle enzymes in the insertion mutants showed that inactivation of genes within one CO2 fixation cluster affected expression of genes within the second cluster, revealing a complex regulatory network.     

Regulation of the Calvin cycle for CO2 fixation as an example for general control mechanisms in metabolic cycles.

The theory of a metabolic cycle with the main portion of its intermediates remaining inside the cycle during one turnover has been developed. On this basis, the regulation of the Calvin cycle is analyzed. It is demonstrated that not only the reactions of non-equilibrium enzymes, as the carboxylation of ribulose 1,5-bisphosphate, but reactions that operate close to a thermodynamic equilibrium, especially the reduction of 3-phosphoglycerate and the transketolase reaction can significantly influence the total turnover period in the Calvin cycle. The role of compensating mechanisms in the maintenance of the photosynthesis rate upon changes of environmental conditions and of enzyme contents is analyzed for the Calvin cycle. It is shown that the change of the total quantity of the metabolites is one of the main self-regulated mechanisms in the Calvin cycle. A change of the ATP/ADP ratio can be used by the cell to maintain the CO2 assimilation rate, when the total quantity of the metabolites is changed. The developed analysis permits to explain some experimental data obtained with transgenic plants with restricted efflux of carbon from the chloroplasts.     

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.     

Wide range of metabolic adaptations to the acquisition of the Calvin cycle revealed by comparison of microbial genomes

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO₂ as a potential substrate for biorefining. The most common CO₂ fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO₂ concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indicate yet unknown regulatory mechanisms in Calvin cycle-utilizing microbes. Our gene ranking provides targets for experiments seeking to improve CO₂ fixation, or engineer novel CO₂-fixing organisms.     

Discovery of the canonical Calvin–Benson cycle

It has been 65 years since the Calvin–Benson cycle was first formulated. In this paper, the development of the concepts that are critical to the cycle is traced and the contributions of Calvin, Benson, and Bassham are discussed. Some simplified views often found in text books such as ascending paper chromatography and the use of the “lollipop” for short labeling are discussed and further details given. Key discoveries that underpinned elucidation of the cycle such as the importance of sedoheptulose phosphate and ribulose 1,5-bisphosphate are described. The interchange of ideas between other researchers working on what is now called the pentose phosphate pathway and the development of the ideas of Calvin and Benson are explored while the gluconeogenic aspects of the cycle are emphasized. Concerns raised about anomalies of label distribution in glucose are considered. Other carbon metabolism pathways associated with the Calvin–Benson cycle are also described. Finally, there is a section describing the rift between Calvin and Benson.

     

Effects of the Calvin cycle on nicotinamide adenine dinucleotide concentrations and redox balances of Xanthobacter flavus

The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in Xanthobacter flavus during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH.     

Spatiotemporal dynamics of the Calvin cycle: multistationarity and symmetry breaking instabilities

The possibility of controlling the Calvin cycle has paramount implications for increasing the production of biomass. Multistationarity, as a dynamical feature of systems, is the first obvious candidate whose control could find biotechnological applications. Here we set out to resolve the debate on the multistationarity of the Calvin cycle. Unlike the existing simulation-based studies, our approach is based on a sound mathematical framework, chemical reaction network theory and algebraic geometry, which results in provable results for the investigated model of the Calvin cycle in which we embed a hierarchy of realistic kinetic laws. Our theoretical findings demonstrate that there is a possibility for multistationarity resulting from two sources, homogeneous and inhomogeneous instabilities, which partially settle the debate on multistability of the Calvin cycle. In addition, our tractable analytical treatment of the bifurcation parameters can be employed in the design of validation experiments.     

Investigating the role of the thiol-regulated enzyme sedoheptulose-1,7-bisphosphatase in the control of photosynthesis

Sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) catalyses the dephosphorylation of sedoheptulose-1,7-bisphosphate in the regenerative phase of the Calvin cycle. Antisense plants with reduced levels of SBPase have decreased photosynthetic capacity and altered carbohydrate status, leading to modifications in growth and development. The catalytic activity of SBPase is regulated by light via the ferredoxin/thioredoxin system. Recently, the amino acids within the SBPase protein involved in this regulatory mechanism have been identified and a deregulated, permanently active form of the enzyme has been produced using site-directed mutagenesis. This paper explores how transgenic Nicotiana tabacum cv. Samsun plants, containing the deregulated form of the SBPase enzyme, may lead to a better understanding of the in vivo role of light activation of this important Calvin cycle enzyme.     

Interruption of the Calvin cycle inhibits the repair of Photosystem II from photodamage

In photosynthetic organisms, impairment of the activities of enzymes in the Calvin cycle enhances the extent of photoinactivation of Photosystem II (PSII). We investigated the molecular mechanism responsible for this phenomenon in the unicellular green alga Chlamydomonas reinhardtii. When the Calvin cycle was interrupted by glycolaldehyde, which is known to inhibit phosphoribulokinase, the extent of photoinactivation of PSII was enhanced. The effect of glycolaldehyde was very similar to that of chloramphenicol, which inhibits protein synthesis de novo in chloroplasts. The interruption of the Calvin cycle by the introduction of a missense mutation into the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) also enhanced the extent of photoinactivation of PSII. In such mutant 10-6C cells, neither glycolaldehyde nor chloramphenicol has any additional effect on photoinactivation. When wild-type cells were incubated under weak light after photodamage to PSII, the activity of PSII recovered gradually and reached a level close to the initial level. However, recovery was inhibited in wild-type cells by glycolaldehyde and was also inhibited in 10-6C cells. Radioactive labelling and Northern blotting demonstrated that the interruption of the Calvin cycle suppressed the synthesis de novo of chloroplast proteins, such as the D1 and D2 proteins, but did not affect the levels of psbA and psbD mRNAs. Our results suggest that the photoinactivation of PSII that is associated with the interruption of the Calvin cycle is attributable primarily to the inhibition of the protein synthesis-dependent repair of PSII at the level of translation in chloroplasts.     

Interruption of the Calvin cycle inhibits the repair of Photosystem II from photodamage

In photosynthetic organisms, impairment of the activities of enzymes in the Calvin cycle enhances the extent of photoinactivation of Photosystem II (PSII). We investigated the molecular mechanism responsible for this phenomenon in the unicellular green alga Chlamydomonas reinhardtii. When the Calvin cycle was interrupted by glycolaldehyde, which is known to inhibit phosphoribulokinase, the extent of photoinactivation of PSII was enhanced. The effect of glycolaldehyde was very similar to that of chloramphenicol, which inhibits protein synthesis de novo in chloroplasts. The interruption of the Calvin cycle by the introduction of a missense mutation into the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) also enhanced the extent of photoinactivation of PSII. In such mutant 10-6C cells, neither glycolaldehyde nor chloramphenicol has any additional effect on photoinactivation. When wild-type cells were incubated under weak light after photodamage to PSII, the activity of PSII recovered gradually and reached a level close to the initial level. However, recovery was inhibited in wild-type cells by glycolaldehyde and was also inhibited in 10-6C cells. Radioactive labelling and Northern blotting demonstrated that the interruption of the Calvin cycle suppressed the synthesis de novo of chloroplast proteins, such as the D1 and D2 proteins, but did not affect the levels of psbA and psbD mRNAs. Our results suggest that the photoinactivation of PSII that is associated with the interruption of the Calvin cycle is attributable primarily to the inhibition of the protein synthesis-dependent repair of PSII at the level of translation in chloroplasts.     

A Mathematical Model of the Calvin Cycle: Analysis of the Steady State

A complete steady-state solution of a recently published mathematicalmodel of the Calvin photosynthesis cycle is obtained in termsof the rate constants of the cycle and certain conservationrules. By means of analytical and numerical tests, the steadystate is shown to be effectively stable over a wide range ofinitial conditions and parameter values. Other properties ofthe steady state, such as its variation with the rate of carbondioxide fixation, are also discussed. Mathematical model, Calvin cycle, photosynthesis, steady state, stability analysis     

Improving carbon fixation pathways

A recent resurgence in basic and applied research on photosynthesis has been driven in part by recognition that fulfilling future food and energy requirements will necessitate improvements in crop carbon-fixation efficiencies. Photosynthesis in traditional terrestrial crops is being reexamined in light of molecular strategies employed by photosynthetic microbes to enhance the activity of the Calvin cycle. Synthetic biology is well-situated to provide original approaches for compartmentalizing and enhancing photosynthetic reactions in a species independent manner. Furthermore, the elucidation of alternative carbon-fixation routes distinct from the Calvin cycle raises possibilities that novel pathways and organisms can be utilized to fix atmospheric carbon dioxide into useful materials.     

Current views on the regulation of autotrophic carbon dioxide fixation via the Calvin cycle in bacteria

The Calvin cycle of carbon dioxide fixation constitutes a biosynthetic pathway for the generation of (multi-carbon) intermediates of central metabolism from the one-carbon compound carbon dioxide. The product of this cycle can be used as a precursor for the synthesis of all components of cell material. Autotrophic carbon dioxide fixation is energetically expensive and it is therefore not surprising that in the various groups of autotrophic bacteria the operation of the cycle is under strict metabolic control. Synthesis of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase, the two enzymes specifically involved in the Calvin cycle, is regulated via end-product repression. In this control phosphoenolpyruvate most likely has an alarmone function. Studies of the enzymes isolated from various sources have indicated that phosphoribulokinase is the target enzyme for the control of the rate of carbon dioxide fixation via the Calvin cycle through modulation of existing enzyme activity. In general, this enzyme is strongly activated by NADH, whereas AMP and phosphoenol-pyruvate are effective inhibitors. Recent studies of phosphoribulokinase inAlcaligenes eutrophus suggest that this enzyme may also be regulated via covalent modification.     

Computer modelling and experimental evidence for two steady states in the photosynthetic Calvin cycle.

We present observations of photosynthetic carbon dioxide assimilation, and leaf starch content from genetically modified tobacco (Nicotiana tabacum) plants in which the activity of the Calvin cycle enzyme, sedoheptulose-1,7-bisphosphatase, is reduced by an antisense construct. The measurements were made on leaves of varying ages and used to calculate the flux control coefficients of sedoheptulose-1,7-bisphosphatase over photosynthetic assimilation and starch synthesis. These calculations suggest that control coefficients for both are negative in young leaves, and positive in mature leaves. This behaviour is compared to control coefficients obtained from a detailed computer model of the Calvin cycle. The comparison demonstrates that the experimental observations are consistent with bistable behaviour exhibited by the model, and provides the first experimental evidence that such behaviour in the Calvin cycle occurs in vivo as well as in silico.     

The isotope effect in the glycine dehydrogenase reaction is the cause of the intramolecular isotope inhomogeneity of glucose carbon of starch synthesized during photorespiration

The isotope distribution of glucose-6-phosphate in the main pathways of its biosynthesis (in the processes of CO2 assimilation and photorespiration in the Calvin cycle and during resynthesis from the degradation products of lipids and proteins) was analyzed. For reconstructing the isotope distribution of glucoso-6-phosphate synthesized in the Calvin cycle during photorespiration, the functioning of the cycle with regard to its coupling with the glycolate chain, which together constitute the photorespiration chain, was considered. In the glycine dehydrogenase reaction of the glycolate cycle, there arises an isotope effect, which determines the distribution of isotopes in the glucose-6-phosphate and other photorespiration products. The isotope effect of the glycine dehydrogenase reaction increases at the expense of the exhaustion of glucose resources feeding the photorespiration chain. As a result, atoms C-3 and C-4 of glucose become enriched with the heavy isotope, and subsequent mixing of atoms and the specificity of interactions in the photorespiration chain lead to an isotope weighting of the other atoms and an uneven distribution of carbon isotopes in glucose-6-phosphate and other photorespiration products. A comparison of the glucose-6-phosphate isotope patterns in different pathways of the synthesis with the experimental data on the distribution of carbon isotopes in starch glucose of storing plant organs led to the conclusion that the starch resources are predominantly formed at the expense of glucose-6-phosphate of photorespiration. This is consistent with the earlier observed enhancement of photorespiration at the stage of plant maturation.     

The Calvin cycle revisited

The sequence of reactions in the Calvin cycle, and the biochemical characteristics of the enzymes involved, have been known for some time. However, the extent to which any individual enzyme controls the rate of carbon fixation has been a long standing question. Over the last 10 years, antisense transgenic plants have been used as tools to address this and have revealed some unexpected findings about the Calvin cycle. It was shown that under a range of environmental conditions, the level of Rubisco protein had little impact on the control of carbon fixation. In addition, three of the four thioredoxin regulated enzymes, FBPase, PRKase and GAPDH, had negligible control of the cycle. Unexpectedly, non-regulated enzymes catalysing reversible reactions, aldolase and transketolase, both exerted significant control over carbon flux. Furthermore, under a range of growth conditions SBPase was shown to have a significant level of control over the Calvin cycle. These data led to the hypothesis that increasing the amounts of these enzymes may lead to an increase in photosynthetic carbon assimilation. Remarkably, photosynthetic capacity and growth were increased in tobacco plants expressing a bifunctional SBPase/FBPase enzyme. Future work is discussed which will further our understanding of this complex and important pathway, particularly in relation to the mechanisms that regulate and co-ordinate enzyme activity.This revised version was published online in October 2005 with corrections to the Cover Date.     

Dependence of the Calvin cycle activity on kinetic parameters for the interaction of non-equilibrium cycle enzymes with their substrates

Kinetic model studies and control analyses of the Calvin photosynthesis cycle have been performed to characterize the dependence of the cycle activity on maximum velocities and Km values for the interaction of the non-equilibrium cycle enzymes and ATP synthetase with their substrates under conditions of light and carbon dioxide saturation. The results show that Km values have no major influence on the cycle activity at optimal concentrations of external orthophosphate. The maximum cycle activity is controlled mainly by the catalytic capacities of ATP synthetase and sedoheptulose-bisphosphatase, and is close to the maximum cycle flux that can be supported by these two enzymes.     

In Situ Association of Calvin Cycle Enzymes,Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase,Ferredoxin-NADP+ Reductase,and Nitrite Reductase with Thylakoid and Pyrenoid Membranes of Chlamydomonas reinhardtii Chloroplasts as Revealed by Immunoelectron Microscopy

The in situ localization of the chloroplast enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco), Rubisco activase, ribose-5-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase was studied by immunoelectron microscopy in Chlamydomonas reinhardtii. Immunogold labeling revealed that, despite Rubisco in the pyrenoid matrix, Calvin cycle enzymes, Rubisco activase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase are associated predominantly with chloroplast thylakoid membranes and the inner surface of the pyrenoid membrane. This is in accord with previous enzyme localization studies in higher plants (K.H. Suss, C. Arkona, R. Manteuffel, K. Adler [1993] Proc Natl Acad Sci USA 90: 5514-5518). Pyrenoid tubules do not contain these enzymes. The pyrenoid matrix consists of Rubisco but is devoid of the other photosynthetic enzymes investigated. Evidence for the occurrence of two Rubisco forms differing in their spatial localization has also been obtained: Rubisco form I appears to be membrane associated like other Calvin cycle components, whereas Rubisco form II is confined to the pyrenoid matrix. It is proposed that enzyme form I represents an active Rubisco when assembled into Calvin cycle enzyme complexes, whereas Rubisco form II may be part of a CO2-concentrating mechanism. Pyrenoidal Calvin cycle complexes are thought to be highly active in CO2 fixation and important for the synthesis of starch around the pyrenoid.     

根据光合作用能量代谢测定光呼吸的方法研究

本文根据光合作用和光呼吸途径能量代谢,通过改变外界CO2和O2浓度,计算卡尔文循环固定的CO2和光呼吸消耗的O2。结果表明,可以通过3种方法计算。方法1,测定在CO2饱和点（A）和正常CO2（A＇）浓度下吸收的CO2,得出光呼吸消耗的O2为：18／19（A-A＇）,卡尔文循环固定的CO2为：1／19（6A＋13A＇＋19Rd）。方法2,测定在不含O2的空气中（O）和正常O2（O’）浓度下释放的O2,得出光呼吸消耗的O2为：-13／5O-O＇-18／5Rd,卡尔文循环固定的CO2为：13／18（O＇—O）。方法3,测定在正常情况下吸收的CO2（A）和释放的O2（O＇）,得出光呼吸消耗的O2为：18（O＇—A＇）,卡尔文循环固定的CO2为：6O＇-5A＇＋Rd。测定在CO2饱和点和正常CO2浓度下吸收的CO2计算出水稻光呼吸释放的CO2占光合作用固定的24％-40％。