γ-6-Phosphogluconolactone, a Byproduct of the Oxidative Pentose Phosphate Pathway, Contributes to AMPK Activation through Inhibition of PP2A.

The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.

Regulation of Rubisco activity by interaction with chloroplast metabolites.

Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-d-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-d-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e. competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.

Photosynthetic Phosphoribulokinase Structures: Enzymatic Mechanisms and the Redox Regulation of the Calvin-Benson-Bassham Cycle.

The Calvin-Benson-Bassham (CBB) cycle is responsible for CO2 assimilation and carbohydrate production in oxyphototrophs. Phosphoribulokinase (PRK) is an essential enzyme of the CBB cycle in photosynthesis, catalyzing ATP-dependent conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-bisphosphate. The oxyphototrophic PRK is redox-regulated and can be further regulated by reversible association with both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidized chloroplast protein CP12. The resulting GAPDH/CP12/PRK complex is central in the regulation of the CBB cycle; however, the PRK-CP12 interface in the recently reported cyanobacterial GAPDH/CP12/PRK structure was not well resolved, and the detailed binding mode of PRK with ATP and Ru5P remains undetermined, as only apo-form structures of PRK are currently available. Here, we report the crystal structures of cyanobacterial (Synechococcus elongatus) PRK in complex with ADP and glucose-6-phosphate and of the Arabidopsis (Arabidopsis thaliana) GAPDH/CP12/PRK complex, providing detailed information regarding the active site of PRK and the key elements essential for PRK-CP12 interaction. Our structural and biochemical results together reveal that the ATP binding site is disrupted in the oxidized PRK, whereas the Ru5P binding site is occupied by oxidized CP12 in the GAPDH/CP12/PRK complex. This structure-function study greatly advances the understanding of the reaction mechanism of PRK and the subtle regulations of redox signaling for the CBB cycle.

The Arabidopsis Plastidial Glucose-6-Phosphate Transporter GPT1 is Dually Targeted to Peroxisomes via the Endoplasmic Reticulum.

Studies on Glucose-6-phosphate (G6P)/phosphate translocator isoforms GPT1 and GPT2 reported the viability of Arabidopsis (Arabidopsis thaliana) gpt2 mutants, whereas heterozygous gpt1 mutants exhibited a variety of defects during fertilization/seed set, indicating that GPT1 is essential for this process. Among other functions, GPT1 was shown to be important for pollen and embryo-sac development. Because our previous work on the irreversible part of the oxidative pentose phosphate pathway (OPPP) revealed comparable effects, we investigated whether GPT1 may dually localize to plastids and peroxisomes. In reporter fusions, GPT2 localized to plastids, but GPT1 also localized to the endoplasmic reticulum (ER) and around peroxisomes. GPT1 contacted two oxidoreductases and also peroxins that mediate import of peroxisomal membrane proteins from the ER, hinting at dual localization. Reconstitution in yeast (Saccharomyces cerevisiae) proteoliposomes revealed that GPT1 preferentially exchanges G6P for ribulose-5-phosphate (Ru5P). Complementation analyses of heterozygous +/gpt1 plants demonstrated that GPT2 is unable to compensate for GPT1 in plastids, whereas GPT1 without the transit peptide (enforcing ER/peroxisomal localization) increased gpt1 transmission significantly. Because OPPP activity in peroxisomes is essential for fertilization, and immunoblot analyses hinted at the presence of unprocessed GPT1-specific bands, our findings suggest that GPT1 is indispensable in both plastids and peroxisomes. Together with its G6P-Ru5P exchange preference, GPT1 appears to play a role distinct from that of GPT2 due to dual targeting.

The transcriptional regulator RbcR controls ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes in the cyanobacterium Synechocystis sp. PCC 6803.

Oxygenic photosynthesis evolved in cyanobacteria, primary producers of striking ecological importance. Like plants, cyanobacteria use the Calvin-Benson-Bassham cycle for CO2 fixation, fuelled by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). In a competitive reaction this enzyme also fixes O2 which makes it rather ineffective. To mitigate this problem, cyanobacteria evolved a CO2 concentrating mechanism (CCM) to pool CO2 in the vicinity of RuBisCO. However, the regulation of these carbon (C) assimilatory systems is understood only partially. Using the model Synechocystis sp. PCC 6803 we characterized an essential LysR-type transcriptional regulator encoded by gene sll0998. Transcript profiling of a knockdown mutant revealed diminished expression of several genes involved in C acquisition, including rbcLXS, sbtA and ccmKL encoding RuBisCO and parts of the CCM, respectively. We demonstrate that the Sll0998 protein binds the rbcL promoter and acts as a RuBisCO regulator (RbcR). We propose ATTA(G/A)-N5 -(C/T)TAAT as the binding motif consensus. Our data validate RbcR as a regulator of inorganic C assimilation and define the regulon controlled by it. Biological CO2 fixation can sustain efforts to reduce its atmospheric concentrations and is fundamental for the light-driven production of chemicals directly from CO2 . Information about the involved regulatory and physiological processes is crucial to engineer cyanobacterial cell factories.

The plastidial pentose phosphate pathway is essential for postglobular embryo development in Arabidopsis.

Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)-required for purine nucleotide and histidine synthesis-and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

A direct spectropolarimetric assay of arabinose 5-phosphate isomerase.

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

Possible Steps of the Carboxylation of Ribulose-1,5-biphosphate from Intermediates: 2,3-Enediol versus 1,2-Enol.

Ribulose 1,5-bisphosphate (RuBP) undergoes enolization to initiate fixation of atmospheric carbon dioxide in the plant carbon cycle. The known model assumes the binding of RuBP to the Rubisco active site with the subsequent formation of 2,3-enediol (2,3,4-trihydroxypent-2-ene-1,5-diyl diphosphate). In the present study, it is assumed that 1,2-enol (2,3,4-trihydroxypent-1-ene-1,5-diyl diphosphate) can be formed in the enolization step to initiate the carboxylation reaction. We have used Kohn-Sham density functional theory on WB97X-D3/Def2-TZVP levels to compare the reaction barriers in the two ways. We considered the pathways of carboxylation of 1/2-ene (mono/di)ol via the C1 and C2 carbons without taking into account the binding of RuBP to the magnesium ion. Calculations of Gibbs free energies confirm the equal probability of the formation of 2,3-enediol and 1,2-enol. Quantum-chemical modeling of enolization and carboxylation reactions supports the important role of the bridging water molecule and diphosphate groups, which provide proton transfer and lower reaction barriers. The results show that carbon dioxide fixation can occur without a magnesium ion, and binding with C1 can have a lower barrier (~12 kcal/mol) than with C2 (~23 kcal/mol).

Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway.

Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.

On a Possible Way to Increase the Efficiency of Photosynthesis.

The paper briefly describes the evolution of the key enzyme of photosynthesis, RuBisCO. Before the emergence of the reaction of carbon dioxide assimilation via photosynthesis, this protein was involved in the methionine metabolism chain. Possibly, for this reason, the carboxylation reaction catalyzed by enzyme proceeds very slowly. In addition to carboxylation, RuBisCO can simultaneously oxidize ribulose bisphosphate, a substrate to which the fixed CO2 is attached. This, in turn, also reduces the effectiveness of photosynthesis. In this regard, the literature discusses various options for increasing plant productivity by creating new forms of RuBisCO or fundamentally different pathways of carbon dioxide assimilation. In this work, we propose a modification of the carboxylation reaction that makes it possible to avoid photorespiration and thus increase the efficiency of photosynthesis.

Metabolic flux analysis for metabolome data validation of naturally xylose-fermenting yeasts.

BACKGROUND: Efficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts. RESULTS: A metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts. CONCLUSIONS: This study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.

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.

Characterization of the heterooligomeric red-type rubisco activase from red algae.

The photosynthetic CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis-powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca.

A unique structural domain in Methanococcoides burtonii ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts as a small subunit mimic.

The catalytic inefficiencies of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) often limit plant productivity. Strategies to engineer more efficient plant Rubiscos have been hampered by evolutionary constraints, prompting interest in Rubisco isoforms from non-photosynthetic organisms. The methanogenic archaeon Methanococcoides burtonii contains a Rubisco isoform that functions to scavenge the ribulose-1,5-bisphosphate (RuBP) by-product of purine/pyrimidine metabolism. The crystal structure of M. burtonii Rubisco (MbR) presented here at 2.6 Å resolution is composed of catalytic large subunits (LSu) assembled into pentamers of dimers, (L2)5, and differs from Rubiscos from higher plants where LSus are glued together by small subunits (SSu) into hexadecameric L8S8 enzymes. MbR contains a unique 29-amino acid insertion near the C terminus, which folds as a separate domain in the structure. This domain, which is visualized for the first time in this study, is located in a similar position to SSus in L8S8 enzymes between LSus of adjacent L2 dimers, where negatively charged residues coordinate around a Mg2+ ion in a fashion that suggests this domain may be important for the assembly process. The Rubisco assembly domain is thus an inbuilt SSu mimic that concentrates L2 dimers. MbR assembly is ligand-stimulated, and we show that only 6-carbon molecules with a particular stereochemistry at the C3 carbon can induce oligomerization. Based on MbR structure, subunit arrangement, sequence, phylogenetic distribution, and function, MbR and a subset of Rubiscos from the Methanosarcinales order are proposed to belong to a new Rubisco subgroup, named form IIIB.

Knockout of SlSBPASE Suppresses Carbon Assimilation and Alters Nitrogen Metabolism in Tomato Plants.

Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme in the Calvin⁻Benson cycle and has been documented to be important in carbon assimilation, growth and stress tolerance in plants. However, information on the impact of SBPase on carbon assimilation and nitrogen metabolism in tomato plants (Solanum lycopersicum) is rather limited. In the present study, we investigated the role of SBPase in carbon assimilation and nitrogen metabolism in tomato plants by knocking out SBPase gene SlSBPASE using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology. Compared with wild-type plants, slsbpase mutant plants displayed severe growth retardation. Further analyses showed that knockout of SlSBPASE led to a substantial reduction in SBPase activity and as a consequence, ribulose-1,5-bisphosphate (RuBP) regeneration and carbon assimilation rate were dramatically inhibited in slsbpase mutant plants. It was further observed that much lower levels of sucrose and starch were accumulated in slsbpase mutant plants than their wild-type counterparts during the photoperiod. Intriguingly, mutation in SlSBPASE altered nitrogen metabolism as demonstrated by changes in levels of protein and amino acids and activities of nitrogen metabolic enzymes. Collectively, our data suggest that SlSBPASE is required for optimal growth, carbon assimilation and nitrogen metabolism in tomato plants.

Spectacular Oscillations in Plant Isoprene Emission under Transient Conditions Explain the Enigmatic CO2 Response.

Plant isoprene emissions respond to light and temperature similarly to photosynthesis, but CO2 dependencies of isoprene emission and photosynthesis are profoundly different, with photosynthesis increasing and isoprene emission decreasing with increasing CO2 concentration due to reasons not yet understood. We studied isoprene emission, net assimilation rate, and chlorophyll fluorescence under different CO2 and O2 concentrations in the strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides), and used rapid changes in ambient CO2 or O2 concentrations or light level to induce oscillations. As isoprene-emitting species support very high steady-state chloroplastic pool sizes of the primary isoprene substrate, dimethylallyl diphosphate (DMADP), which can mask the effects of oscillatory dynamics on isoprene emission, the size of the DMADP pool was experimentally reduced by either partial inhibition of isoprenoid synthesis pathway by fosmidomycin-feeding or by changes in ambient gas concentrations leading to DMADP pool depletion in intact leaves. In feedback-limited conditions observed at low O2 and/or high CO2 concentration under which the rate of photosynthesis is governed by the limited rate of ATP and NADPH formation due to low chloroplastic phosphate levels, oscillations in photosynthesis and isoprene emission were repeatedly induced by rapid environmental modifications in both partly fosmidomycin-inhibited leaves and in intact leaves with in vivo reduced DMADP pools. The oscillations in net assimilation rate and isoprene emission in feedback-inhibited leaves were in the same phase, and relative changes in the pools of photosynthetic metabolites and DMADP estimated by in vivo kinetic methods were directly proportional through all oscillations induced by different environmental perturbations. We conclude that the oscillations in isoprene emission provide direct experimental evidence demonstrating that the response of isoprene emission to changes in ambient gas concentrations is controlled by the chloroplastic reductant supply.

Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified.

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes. METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants. RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis. CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity. GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA(+) (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-Å crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA(+) protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO(2) uptake and biomass production by photosynthetic organisms.

6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.

D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg2+ and Mn2+ and displayed similar K' values (56-75 µM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V max (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg2+ and Mn2+, and displayed similar thermostabilities. The PFKs have similar K m values for fructose 6-phosphate (0.61-0.94 µM) and for ATP (0.38-0.82 µM), while Pfk1-MGA3 had a 2-fold lower V max (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.