Thioredoxin activation of phosphoribulokinase in a bi-enzyme complex from Chlamydomonas reinhardtii chloroplasts

The activation of oxidized phosphoribulokinase either "free" or as part of a bi-enzyme complex by reduced thioredoxins during the enzyme reaction was studied. In the presence of reduced thioredoxin, the product of the reaction catalyzed by phosphoribulokinase within the bi-enzyme complex does not appear in a linear fashion. It follows a mono-exponential pattern that suggests a slow dissociation process of the bi-enzyme complex in the assay cuvette. A plot of the steady state of product appearance against thioredoxin concentration gave a sigmoid curve. On the basis of our experimental results, we propose a minimum model of the activation of phosphoribulokinase by reduced thioredoxin. Reduced thioredoxin may act on the phosphoribulokinase, either within the complex or in the dissociated metastable form. However, the time required to activate the enzyme as part of the complex is shorter (about 20 s) than that required to activate the dissociated form (about 10 min). This might be of physiological relevance, and we discuss the role of the interactions between phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase in the regulation of the Calvin cycle.     

Regulation of chloroplast phosphoribulokinase by the ferredoxin/thioredoxin system

A newly found form of chloroplast phosphoribulokinase (designated the “regulatory form”) required reduced thioredoxin for activity. A second form of the enzyme (the “nonregulatory form”) was not appreciably affected by thioredoxin. The thioredoxin required for activation of the regulatory enzyme could be reduced (i) photochemically by chloroplast membranes that were supplemented with ferredoxin and ferredoxin-thioredoxin reductase or (ii) chemically in the dark with the sulfhydryl reagent dithiothreitol. Following activation by reduced thioredoxin, phosphoribulokinase was deactivated by the soluble chloroplast oxidants dehydroascorbate and oxidized glutathione. The results suggest that the regulatory form of phosphoribulokinase resembles fructose 1,6-bisphosphatase in its mode of regulation by the ferredoxin/thioredoxin system.     

Concerted complex assembly and GTPase activation in the chloroplast signal recognition particle

The universally conserved signal recognition particle (SRP) and SRP receptor (SR) mediate the cotranslational targeting of proteins to cellular membranes. In contrast, a unique chloroplast SRP in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll a/b binding (LHC) proteins. In both pathways, dimerization and activation between the SRP and SR GTPases mediate the delivery of cargo; whether and how the GTPase cycle in each system adapts to its distinct substrate proteins were unclear. Here, we show that interactions at the active site essential for GTPase activation in the chloroplast SRP and SR play key roles in the assembly of the GTPase complex. In contrast to their cytosolic homologues, GTPase activation in the chloroplast SRP-SR complex contributes marginally to the targeting of LHC proteins. These results demonstrate that complex assembly and GTPase activation are highly coupled in the chloroplast SRP and SR and suggest that the chloroplast GTPases may forego the GTPase activation step as a key regulatory point. These features may reflect adaptations of the chloroplast SRP to the delivery of their unique substrate protein.     

Dual effects of organic solvents on chloroplast phosphoribulokinase and NADP-glyceraldehyde-3-P dehydrogenase

Organic solvents miscible in water (cosolvents) exerted a dual effect on the activation stage of two thioredoxin-linked enzymes of the reductive pentose phosphate cycle, phosphoribulokinase and NADP-glyceraldehyde-3-P dehydrogenase, both from spinach chloroplast; the enzyme specific activity was stimulated and inhibited by low and high concentrations of alcohols, respectively. On the contrary, cosolvents inhibited the catalytic process. In the stimulation of phosphoribulokinase activation, organic solvents reduced the requirement for thioredoxin-f and changed the thiol specificity, so that monothiols became functional. The cosolvent-mediated enhancement of NADP-glyceraldehyde-3-P dehydrogenase was obtained in the absence of modulators. With both enzymes, the concentration of the organic solvents required for activation was inversely proportional to its hydrophobicity (1-butanol < 1-propanol < 2-propanol < ethanol). The present results demonstrate the participation of a new component, the enzyme microenvironment, in the regulation of thioredoxin-linked chloroplast enzymes.     

Symmetry-regulated dynamics of multi-enzyme complexes. A model of a pyruvate dehydrogenase complex from Escherichia coli

A dynamic model for quaternary structure of a multienzyme complex is considered. The model is based on the supposition of simultaneously existing similar subunits in a number of different conformational states in the "core" of the multienzyme complex. It is supposed that cyclic conformational transitions of the "core" subunits conserve the symmetry of the entire complex. Such transitions drive the core dynamics as well as the suprastructural multienzyme dynamics. The dynamic model is constructed for the pyruvate dehydrogenase complex from E. coli in a supposition of three different conformers existing in its "core" which correspond to the three steps of the cyclic catalytic process. The model is in accordance with the data from the literature.     

Influence of the activation status and of ATP on phosphoribulokinase degradation

The light-regulated chloroplast enzyme phosphoribulokinase (EC 2.7.1. 19) exists in two forms. In darkness this enzyme is present in an oxidized form, which is inactive. It is activated in the light by a thioredoxin-mediated reduction. In extracts from young wheat leaves (Triticum aeestivum L.) phosphoribulokinase as well as some other thioredoxin-modulated enzymes can be activated by the artificial reductant dithiothreitol (DTT). The influence of the activation status and of the substrate ATP on phosphoribulokinase stability was investigated in the presence of endogenous endopeptidases from senescing wheat leaves. Similar experiments were performed with purified phosphoribulokinase from spinach in the presence of exogenous, purified endopeptidases (chymotrypsin and trypsin). Phosphoribulokinase stability was analysed by immunoblotting and activity measurements. Both systems led to similar conclusions. DTT (reductant and ATP (substrate) stabilized phosphoribulokinase in wheat leaf extracts as well as partially purified phosphoribulokinase from spinach. The combination of both effectors was far more protective than either effector alone. DTT had hardly any effect on the degradation of thioredoxin-independent chloroplast enzymes such as glutamate synthase and glutamine synthetase. These results suggest that the activation status and substrate concentrations are not only important for the activity of phosphoribulokinase, but are also relevant for the susceptibility of this enzyme to proteolysis.     

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer. Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques. Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex. Full activation is achieved in the slow isomerization reaction. In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction. These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.     

Inhibition of spinach phosphoribulokinase by DL-glyceraldehyde.

Spinach chloroplast phosphoribulokinase is inhibited by DL-glyceraldehyde. The inhibition is non-competitive with respect to ribulose 5-phosphate (Ki 19mM) and ATP (Ki 20mM). The inhibition is discussed in relation to a previously reported inhibition of CO2 assimilation in intact and envelope-free chloroplasts by DL-glyceraldehyde. It is concluded that the inhibition of phosphoribulokinase is insufficient to account for the inhibition, by DL-glyceraldehyde, of O2 evolution with ribose 5-phosphate as substrate and that a further site of inhibition is also present in this system.     

Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores

Lipolysis is the biochemical pathway responsible for the catabolism of triacylglycerol (TAG) stored in cellular lipid droplets. The hydrolytic cleavage of TAG generates non-esterified fatty acids, which are subsequently used as energy substrates, essential precursors for lipid and membrane synthesis, or mediators in cell signaling processes. Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, it is most abundant, however, in white and brown adipose tissue. Over the last 5years, important enzymes and regulatory protein factors involved in lipolysis have been identified. These include an essential TAG hydrolase named adipose triglyceride lipase (ATGL) [annotated as patatin-like phospholipase domain-containing protein A2], the ATGL activator comparative gene identification-58 [annotated as α/β hydrolase containing protein 5], and the ATGL inhibitor G0/G1 switch gene 2. Together with the established hormone-sensitive lipase [annotated as lipase E] and monoglyceride lipase, these proteins constitute the basic "lipolytic machinery". Additionally, a large number of hormonal signaling pathways and lipid droplet-associated protein factors regulate substrate access and the activity of the "lipolysome". This review summarizes the current knowledge concerning the enzymes and regulatory processes governing lipolysis of fat stores in adipose and non-adipose tissues. Special emphasis will be given to ATGL, its regulation, and physiological function.     

Non-redox protein interactions in the thioredoxin activation of chloroplast enzymes.

Thioredoxin derivatives lacking SH groups such as S,S'-dicarboxymethyl-, dicarboxamidomethyl-thioredoxin and cysteine----serine mutant protein are capable of activating chloroplast NADP malate dehydrogenase and fructose-bisphosphatase when added to enzyme assays together with suboptimal amounts of native thioredoxin. The modified thioredoxins alone are inactive. These findings indicate that protein-protein interactions play a significant role in addition to disulfide/thiol exchange reactions in the light-driven regulation of plant enzymes by the various plant thioredoxins.     

Complement activation by a univalent hapten-antibody complex.

The univalent hapten, nonadeca lysyl epsilon-Dnp-lysine, binds tightly to rabbit anti-2,4-dinitrophenyl antibody, and the complex has a sedimentation coefficient of 6.7, characteristic of a single antibody molecule. In this communication, we show that this complex is a good activator of the serum complement system. For activation to occur, the univalent hapten must contain the specific group which binds to the antibody, and also the polycationic chain. In addition, activation requires a functional complement-binding region on the intact antibody molecule. The classical pathway appears to be involved since the first, fourth, and second components of complement are markedly depleted when the complement system is activated by this univalent hapten-antibody complex.     

Component X. An immunologically distinct polypeptide associated with mammalian pyruvate dehydrogenase multi-enzyme complex

The mammalian pyruvate dehydrogenase multi-enzyme complex contains a tightly-associated 50 000-Mr polypeptide of unknown function (component X) in addition to its three constituent enzymes, pyruvate dehydrogenase (E1), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3) which are jointly responsible for production of CoASAc and NADH. The presence of component X is apparent on sodium dodecyl sulphate/polyacrylamide gel analysis of the complex, performed in Tris-glycine buffers although it co-migrates with the E3 subunit on standard phosphate gels run under denaturing conditions. Refined immunological techniques, employing subunit-specific antisera to individual components of the pyruvate dehydrogenase complex, have demonstrated that protein X is not a proteolytic fragment of E2 (or E3) as suggested previously. In addition, anti-X serum elicits no cross-reaction with either subunit of the intrinsic kinase of the pyruvate dehydrogenase complex. Immune-blotting analysis of SDS extracts of bovine, rat and pig cell lines and derived subcellular fractions have indicated that protein X is a normal cellular component with a specific mitochondrial location. It remains tightly-associated with the 'core' enzyme, E2, on dissociation of the complex at pH 9.5 or by treatment with 0.25 M MgCl2. This polypeptide is not released to any significant extent from E2 by p-hydroxymercuriphenyl sulphonate, a reagent which promotes dissociation of the specific kinase of the complex from the 'core' enzyme. Incubation of the complex with [2-14C]pyruvate in the absence of CoASH promotes the incorporation of radio-label, probably in the form of acetyl groups, into both E2 and component X.     

Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

The light‐dependent regulation of stromal enzymes by thioredoxin (Trx)‐catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx‐mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol‐dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx‐linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de‐epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox‐controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.     

A supramolecular light-harvesting complex from chloroplast photosystem-II membranes.

In this study, we report on the composition of a photosystem-II antenna preparation which contains three chlorophyll-a/b proteins (CP), CP29, CP24 and light-harvesting complex (LHC) II obtained from Zea mays grana membranes as previously described [Dainese, P. & Bassi, R. (1991) J. Biol. Chem. 266, 8136-8142]. We demonstrate that the three chlorophyll proteins are present in the preparation with a 3:3:9 molar ratio and that they form a supramolecular antenna complex which represents one third of the photosystem-II antenna system. Phosphorylation experiments show that this complex is involved in the mechanism of regulation of excitation-energy distribution between photosystems: phosphorylation of the membranes induces dissociation of the LHCII moiety from the CP29-CP24 moiety and changes in the aggregation state of LHCII components of the CP29-CP24-LHCII complex. The LHCII subpopulations of the complex are shown to be distinct from the total LHCII population by isoelectrofocusing analysis. On the basis of these data and in the light of the stoichiometry of photosystem-II chlorophyll-binding proteins, we propose a model for the organization of photosystem-II antenna system.     

Molecular approaches to probe differential NADH activation of phosphoribulokinase isozymes from Rhodobacter sphaeroides

The cbbPI and cbbPII genes from Rhodobacter sphaeroides, encoding highly similar phosphoribulokinase (PRK) isozymes, PRK I and PRK II, respectively, exhibited differential allosteric activation by NADH. The two cbbP genes were cloned into expression vectors and homogeneous recombinant protein prepared. PRK II was found to be inherently less stable than PRK I; however, the addition of substrate ATP resulted in the complete protection of both isozymes to a 15-min incubation at 50 degrees C. The relative molecular masses for both octameric isozymes were determined to be approximately 230,000; however, the protective effect of ATP was in accordance with aggregation of monomers to a molecular mass of approximately 750,000. While PRK I exhibited a nearly absolute dependence upon NADH for activity, PRK II retained substantial activity in the absence of NADH. PRK chimeras were thus constructed to facilitate elucidation of the basis for the differential effect of NADH, with advantage taken of the relative sequence identity of about 90% between the two isozymes. Chimeras were constructed either by in vivo homologous recombination, using the sacB gene from Bacillus subtilis as a conditionally lethal marker, or by using convenient restriction sites to combine different parts of the two cbbP genes. The PRK chimeras generated contained either the amino-terminal domain of PRK II and the carboxy-terminal domain of PRK I or the opposite configuration. Subsequent analyses of the chimeras pointed to particular regions and residue(s) as likely being important for NADH activation.     

The oxygen-evolving complex of chloroplast membranes

The oxygen-evolving complex (OEC) of plants is the main energy-transforming structure of chloroplast membranes, in which light energy is used for photosynthetic oxidation of intracellular water and oxygen formation. The conducted research has resulted in isolation of functionally active OEC of higher plants and elucidation of its molecular composition, photochemical properties and structural organization. The OEC has been revealed to represent the dimer of the pigment-lipoprotein complexes of photosystem 2 (PLPC PS-2) associated in a chloroplast membrane according to the mirror symmetry rule into an integrate structure based on hydrophobic bonds. The model has been developed for the structure of the dimeric complex of PS-2 that has the function of oxygen formation. This model was confirmed by the X-ray analysis of crystals of the dimeric complex of PS-2. The concept about the fact that the “hydrophobic boiler” determining the formation of the water-oxidizing center of the OEC is formed in the area of association of the reaction centers of monomeric PLPCs PS-2 was advanced based on the regularities of change in the functional activity of the OEC under the action of stress-factors. The new scheme has been advanced for the two-anode organization of the water-oxidizing center as the main condition for realizing the process of molecular oxygen formation. The mechanism of the process of photosynthetic water oxidation and molecular oxygen formation has been developed based on the experimental data about the structural organization of the OEC and its water-oxidizing center. The quantum-chemical modeling of the process showed that its course corresponds to the mechanism suggested.     

Studies on the multi-enzyme complex of yeast fatty-acid synthetase. Reversible dissociation and isolation of two polypeptide chains.

1. The multi-enzyme complex of fatty acid synthetase, Mr 2300,000, was dissociated by acylation with dimethyl maleic anhydride under conditions which lead to an acylation of about 30% of the epsilon amino groups of lysine. The complete dissociation into the subunits alpha and beta is demonstrated by analytical ultracentrifugation as well as disc gel electrophoresis. 2. This dissociation is reversible. Hydrolysis of the resulting protein dicarboxylic acid monoamides under mildly acidic conditions leads to the unmodified subunits, which can be reconstituted to form a complex displaying about 60% of the original activity. 3. The subunits were isolated by sucrose-density-gradient centrifugation and studied for the different partial enzyme activities involved in long-chain fatty acid synthesis: malonyl, palmitoyl and acetyl transferase, enoyl reductase and dehydratase were shown to be exclusive functions of the beta chains of the complex, confirming a pentafunctional role of this subunit.