Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000 x g, followed by 90,000 x g and finally at 150,000 x g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000 x g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000 x g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO2 fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.     

Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000×g, followed by 90,000×g and finally at 150,000×g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000×g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000×g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO₂ fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.     

Photosynthetic Carbon Metabolism in Leaves and Isolated Chloroplasts from Spinach Plants Grown under Short and Intermediate Photosynthetic Periods

Responses of foliar and isolated intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia oleracea cv Wisconsin Bloomsdale) plants exposed to a shortened photosynthetic period (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propagated continuously on a 12-hour light/12-hour dark cycle, 14 to 15 days were required, subsequent to a shift from 12 to 7 hours daylength, for 7-hour plants to begin to grow at rates comparable to those of 12-hour daylength plants. Because of shorter daily durations of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour than in the 12-hour plants. The result was that 7-hour plants established a 1.5- to 2.0-fold higher total PS rate than 12-hour plants.

Intact chloroplasts isolated from the leaves of 7-hour plants (7-h PLD) displayed 1.5- to 2.0-fold higher PS rates than plastids isolated from 12-hour plants (12-h PLD). Plastid lamellae prepared from 7- and 12-h PLD isolates displayed equivalent rates of ferredoxin-dependent ATP and NADPH photoformation indicating that electron transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in leaves as well as intact PLD isolates, of dark to light transitional increases in Calvin cycle intermediates, e.g., ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3-PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6-bisphosphate phosphatase, indicated that 7-hour plant leaves displayed higher PS rates (than 12-hour plants), because there was a higher magnitude of activity of the Calvin cycle.

Although both the foliar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-hour compared with 12-hour plant foliage, the higher 7-hour plant total PS rates indicated that maximal sucrose and starch levels did not mediate any `feedback' inhibition of PS. The higher 7-hour plant foliar and PLD PS rates resulted in higher glucose-1-P levels as well as a higher ratio of 3-PGA:Pi, both factors of which would enhance the activity of chloroplast ADP-glucose pyrophosphorylase, and which were attributed to be causal to the higher starch synthesis rates observed in 7-hour plant foliage and PLD isolates.

     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Non-cyclic photoreductive carbon fixation in photosynthesis. Light and dark transients of the glycerate-3-P special pair

It is demonstrated that carbon fixation in photosynthesis is regulated in two kinetically coupled pathways involving the specialized pair of non-equivalent, enzyme-bound glycerate-3-P (3-PGA) molecules obtained from ribulose 1,5-bisphosphate (RuBP) carboxylation in the light. A non-cyclic pathway is suggested (reaction 2) for the direct biosynthesis of sucrose from the 3-PGA obtained from C-3, C-4 and C-5 of the six-carbon carboxylation adduct. Concomitant to the appearance of sucrose as the principal product, the Mg2+-bound 3-PGA molecule formed from C-1, C-2 and C-2' of the C6 intermediate is released and subsequently reduced in regenerating the RuBP. It is proposed that the nocturnal inhibitor, 2-carboxyarabinitol-1-phosphate (1-PCA) is obtained from a condensation of 3-PGA and glyceraldehyde.     

Photoreductive path of carbon fixation in green plant photosynthesis. Reaction pathway of six-carbon ribulose 1,5-bisphosphate carboxylation adduct intermediate

In this paper we examine the six-carbon intermediate pathway of ribulose 1,5-bisphosphate (RuBP) carboxylation reaction in photosynthesis. Based on the observed reactions of purified RuBP carboxylase, mechanisms are described for carbon dioxide assimilation leading to the hydrolytic splitting of the six-carbon intermediate to two enzyme-bound glycerate-3-P (3-PGA) molecules. It is concluded that, under photosynthetic conditions, the reduction of enzyme-bound NADP+ by the chlorophyll is responsible for the rapid carboxylase turnover rate given by the lifetime, tau L = 0.4 s, which is nearly two orders of magnitude shorter than the corresponding value, tau D = 11 +/- 3 s, for the dark decay of enzyme-bound RuBP. The nocturnal inhibition and photoactivation of RuBP carboxylation are described in terms of the reversible light-dark cycles of the NADP+/NADPH redox couple and endogenous changes that accompany the 2-carboxy-D-arabinitol-1-phosphate binding to the enzyme active site.     

Cascade of bioreactors in series for conversion of 3-phospho-D-glycerate into D-ribulose-1,5-bisphosphate: kinetic parameters of enzymes and operation variables

A novel scheme employing enzymatic catalysts is described enabling conversion of D-ribulose-1,5-bisphosphate (RuBP) from 3-phospho-D-glycerate (3-PGA) without loss of carbon. Bioreactors harboring immobilized enzymes namely, phosphoglycerate kinase (PGK), glycerate phosphate dehydrogenase, triose phosphate isomerase (TIM), aldolase, transketolase (TKL), phosphatase (PTASE/FP), epimerase (EMR) and phosphoribulokinase (PRK), in accordance with this novel scheme were employed. These reactors were designed and constructed based on simulations carried out to study their performance under various operational conditions and allowed production of about 56 +/- 3% RuBP from 3-PGA. This method of synthesis of RuBP from 3-PGA employing immobilized enzyme bioreactors may be used for continuous regeneration of RuBP in biocatalytic carbon dioxide fixation processes from emissions where RuBP acts as acceptor of carbon dioxide to produce 3-PGA, rendering the fixation process continuous.     

Increased activity of the tandem fructose-1,6-bisphosphate aldolase,triosephosphate isomerase and fructose-1,6-bisphosphatase enzymes in <Emphasis Type="Italic">Anabaena</Emphasis> sp. strain PCC 7120 stimulates photosynthetic yield

The regulation of photosynthetic yield at the genetic level has largely focused on manipulation of the catalytic enzymes in the Calvin cycle by genetic engineering. In order to investigate the contribution of increased enzymatic activity in the Calvin cycle on photosynthetic yield, the rice fructose-1,6-bisphosphate aldolase (FBA), spinach triosephosphate isomerase (TPI) and wheat fructose-1,6-bisphosphatase (FBPase) genes were cloned in tandem and co-overexpressed in cyanobacterium Anabaena sp. strain PCC 7120 cells. The enzymatic activities of FBA, TPI and FBPase, as well as sedoheptulose-1,7-bisphosphatase (SBPase), were remarkably increased in transgenic cells relative to the wild-type. The photosynthetic yield, as reflected by photosynthetic O₂ evolution and dry cellular weight, was also markedly increased in transgenic cells versus wide-type cells. The activity of SBPase is considered the most important factor for ribulose-1,5-bisphosphate (RuBP) regeneration in the Calvin cycle, and increased activity of TPI alone in transgenic cells does not stimulate photosynthetic yield. Thus, the increased activity of FBA and FBPase, but not TPI, significantly improved photosynthetic yield in transgenic cells by stimulating SBPase activity and consequently accelerating the RuBP regeneration rate.     

Effect of phosphon-D on photosynthetic light reactions and on reactions of the oxidative and reductive pentose phosphate cycle in a reconstituted spinach (Spinacia oleracea L.) chloroplast system

Phosphon-D (tributyl-2, 4-dichlorobenzylphosphonium chloride), known as an inhibitor of gibberellin biosynthesis, enhances photosynthetic electron transport by up to 200%, with Fe(CN) ₆ ^3- and NADP⁺ being the electron acceptors. Maximum stimulation is reached at phosphon-D concentrations around 2–5 M. At the same time photosynthetic ATP formation is gradually inhibited. Phosphon-D concentrations over 0.1 mM inhibit electron transport. The uncoupling activity of phosphon-D is manifested by inhibition of noncyclic ATP synthesis and by stimulation of light-induced electron flow. The inhibition of ATP synthesis drastically decreases photosynthetic carbon assimilation in a reconstituted spinach chloroplast system. The two ATP-dependent kinase reactions of the reductive pentose phosphate cycle become the rate-limiting steps. On the other hand a stimulated photoelectron transport increases the NADPH/NADP⁺ ratio, resulting in a drastic inhibition of chloroplast glucose-6-phosphate dehydrogenase (EC 1.1.1.49), the key enzyme of the oxidative pentose phosphate cycle. When light-induced electron flow is inhibited by high phosphon-D concentrations and the NADPH/NADP⁺ ratio is low, the light-dependent inhibition of glucose-6-phosphate dehydrogenase is gradually abolished.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - B-Nine N-dimethylaminosuccinamic acid - CCC (2-chloroethyl)-trimethylammonium chloride - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCPIP dichlorophenolindophenol - G-6-PDH glucose-6-phosphate dehydrogenase - FBP fructose bisphosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglyceric acid - Posphon-D tributyl-2,4-dichlorobenzylphosphonium chloride - PMP pentose monophosphates - PPC pentose phosphate cycle - RuBP ribulose bisphosphate - Ru-5-P ribulose-5-phosphate Dedicated to Prof. Dr. Drs.h.c. Adolf Butenandt on the occasion of his 75. birthday     

Limiting Factors in Photosynthesis: VI. Regeneration of Ribulose 1,5-Bisphosphate Limits Photosynthesis at Low Photochemical Capacity

Earlier work (SE Taylor, N Terry [1984] Plant Physiol 75: 82-86) has shown that the rate of photosynthesis may be colimited by photosynthetic electron transport capacity, even at low intercellular CO₂ concentrations. Here we monitored leaf metabolites diurnally and the activities of key Calvin cycle enzymes in the leaves of three treatment groups of sugar beet (Beta vulgaris L.) plants representing three different in vivo photochemical capacities, i.e. Fe-sufficient (control) plants, moderately Fe-deficient, and severely Fe-deficient plants. The results show that the decrease in photosynthesis with Fe deficiency mediated reduction in photochemical capacity was through a reduction in ribulose 1,5-bisphosphate (RuBP) regeneration and not through a decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Based on measurements of ATP and NADPH and triose phosphate/3-phosphoglycerate ratios in leaves, there was little evidence that photosynthesis and RuBP regeneration in Fe-deficient leaves were limited directly by the supply of ATP and NADPH. It appeared more likely that photochemical capacity influenced RuBP regeneration through modulation of enzymes in the photosynthetic carbon reduction cycle between fructose-6-phosphate and RuBP; in particular, the initial activity of ribulose-5-phosphate kinase was strongly diminished by Fe deficiency. Starch and sucrose levels changed independently of one another to some extent during the diurnal period (both increasing in the day and decreasing at night) but the average rates of starch or sucrose accumulation over the light period were each proportional to photochemical capacity and photosynthetic rate.     

Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II

We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO2 fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO2 fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO2 fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO2 also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO2 fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.     

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.     

Dynamic discrete model of flashing light effect in photosynthesis of microalgae

For a photobioreactor for mass-culturing microalgae, it is known that flashing light effect enhances the efficiency of photosynthesis. A dynamic model for photosynthesis was developed to elucidate this effect. A particular feature of the model is that discrete RuBP particles circulate in the Calvin cycle and their speeds in the cycle are determined by the amount of ATP generated in the photon reception process. This can realise the light saturation under continuous light and the flashing light effect under fluctuating illumination. Laboratory experiments were conducted to obtain model parameters by curve-fitting for Chaetoceros calcitrans. The present model demonstrates the light flashing effect moderately well and elucidates its mechanism reasonably.     

RuBisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RuBisCO-like proteins and photosynthetic RuBisCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme in the fixation of CO(2) in the Calvin cycle of plants. Many genome projects have revealed that bacteria, including Bacillus subtilis, possess genes for proteins that are similar to the large subunit of RuBisCO. These RuBisCO homologues are called RuBisCO-like proteins (RLPs) because they are not able to catalyse the carboxylase or the oxygenase reactions that are catalysed by photosynthetic RuBisCO. It has been demonstrated that B. subtilis RLP catalyses the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. The structure of DK-MTP-1-P is very similar to that of ribulose-1,5-bisphosphate (RuBP) and the enolase reaction is a part of the reaction catalysed by photosynthetic RuBisCO. In this review, functional and evolutionary relationships between B. subtilis RLP of the methionine salvage pathway, other RLPs, and photosynthetic RuBisCO are discussed. In addition, the fundamental question, 'How has RuBisCO evolved?' is also considered, and evidence is presented that RuBisCOs evolved from RLPs.     

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.     

Response of Rice Leaves to Low Temperature I. Changes in Basic Biochemical Parameters

When the seedlings of two rice cultivars, IR8 (low-temperaturesensitive) and Somewake (low-temperature tolerant), were exposedto a low temperature of 15?C, the normal increase in the chlorophyllcontent of the developing 4th leaf blade completely ceased whileincreases in protein content continued at a low rate in bothcultivars. Analysis of soluble and insoluble proteins in the4th leaf blade of IR8 by SDS-polyacrylamide gel electrophoresisrevealed that synthesis of RuBP carboxylase and of several thylakoidproteins responsible for photosynthetic electron transport andphotophosphorylation was greatly inhibited at the low temperature.It was also found that increases in the activities of some enzymesof the Calvin cycle, such as RuBP carboxylase, fructose bisphosphataseand NADP-glyceraldehyde-3-P dehydrogenase, as well as of catalase,were specifically inhibited during growth at the low temperature.These results suggest that the synthesis of intracellular components,in particular of key proteins required for photosynthesis, isspecifically susceptible to low-temperature stress during developmentof rice leaves. (Received June 21, 1989; Accepted January 11, 1990)     

A pathway of the autotrophic CO2 fixation in Chloroflexus aurantiacus

Autotrophically grown cells of Chloroflexus aurantiacus B-3 were shown to possess activity of ATP-dependent malate lyase (acetylating CoA). ATP: malate lyase is supposed to be the specific enzyme of the cycle of the autotrophic CO₂ fixation, in which pyruvate synthase, pyruvate phosphate dikinase, phosphoenolpyruvate (PEP) carboxylase and malate dehydrogenase are involved as well. The main product of the CO₂ fixation cycle is glyoxylate, which could further be converted into 3-phosphoglyceric acid (3-PGA) in the reactions of either glycerate or serine pathway. The enzymes of both pathways were detected in C. auratiacus B-3. The results of the in vivo studies of glyxoylate and glycine metabolism, as well as the inhibitor analysis using fluoroacetate (FAc), isonicotinic acid hydrazide (INH), and 4-aminopterin (4-AP) confirm the operation of the proposed pathway in Chloroflexus.Abbreviations 3-PGA 3-phosphoglyceric acid - 4-AP 4-aminopterin - FAc fluoroacetate - INH isonicotinic acid hydrazide - MV methyl viologen - PEP phosphoenolpyruvate - THF tetrahydrofolate - TPP thiamine pyrophosphate     

Enzymes of the autotrophic pathway in mating partners and transconjugants of Nocardia opaca 1 b and Rhodococcus erythropolis

Enzyme activities have been measured in the partners of a bacterial mating system consisting of the hydrogen autotroph Nocardia opaca (donor and Aut^- recipient), the heterotroph Rhodococcus erythropolis (recipient) and intra- and interspecies transconjugants after growth on fructose, pyruvate and under autotrophic conditions. Specific activities of each of the enzymes hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase were high in autotrophically grown cells of the donor and the transconjugants: they amounted to only 10% after growth on pyruvate. The recipient cells did not grow autotrophically and the enzymes mentioned were not detectable even after growth on pyruvate. Other enzymes of the Calvin cycle were constitutively formed in all strains examined.The properties of hydrogenase (K _m for NAD, R_f in gel electrophoresis) and of ribulosebisphosphate carboxylase (K _m for RuBP and R_f) were the same in the donor and transconjugant cells. The properties of glucose-6-phosphate dehydrogenase (K _m for G-6-P and mode of inhibition by ATP and phosphoenolpyruvate) were the same in the recipient and the interspecies transconjugant cells and differed from those of the donor cells. The curves of growth under autotrophic conditions in batch culture of the donor and interspecies transconjugant were almost congruent. The specific activities of hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase increased from 40% at the beginning to 100% at the end of the exponential growth phase; these enzymes were under coordinate control.The results are in accordance with genetic studies: the genetic information for autotrophic growth is localized on a so far unidentified genetic element and is transferred en bloc from N. opaca to Aut^- mutants of the same strain or to recipient bacteria such as R. erythropolis; expression in the wild type and transconjugant cells is the same.Abbreviations G-6-P glucose-6-phosphate - 6-PG 6-phosphogluconate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II

We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO₂ fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO₂ fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO₂ fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO₂ also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO₂ fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.