Anaplerotic flux into the Calvin-Benson cycle. Integration in carbon and energy metabolism of Helianthus annuus

Plants assimilate carbon primarily via the Calvin-Benson cycle. Two companion papers reports evidence for anaplerotic carbon flux into this cycle. To estimate flux rates in Helianthus annuus leaves based on gas exchange measurements, we here expanded Farquhar-von Caemmerer-Berry photosynthesis models by terms accounting for anaplerotic respiration and energy recycling. In line with reported isotope evidence (companion papers), we found relative increases in anaplerotic flux as intercellular CO2 concentrations, Ci, decrease below a change point. At Ci=136 and 202 ppm, we found absolute rates of 2.99 and 2.39 μmol Ru5P m-2 s-1 corresponding to 58.3 and 28.2% of net CO2 assimilation, 13.1 and 10.7% of ribulose 1,5-bisphosphate regeneration, and 22.2 and 15.8% of Rubisco carboxylation (futile carbon cycling), respectively. Anaplerotic respiration governs total day respiration with contributions of 81.3 and 77.6%, and anaplerotic relative to photorespiratory CO2 release amounts to 63.9 and 67%, respectively. Furthermore, anaplerotic flux significantly increases absolute ATP demands and ATP-to-NADPH demand ratios of photosynthesis and may explain increasing sucrose-to-starch carbon partitioning ratios with decreasing Ci. We propose that anaplerotic flux can occur under both Rubisco and RuBP-limited growth conditions. Overall, our work introduces the anaplerotic pathway as central component in carbon and energy metabolism of C3 plants.

Anaplerotic flux into the Calvin-Benson cycle. Isotope evidence for in vivo occurrence in Helianthus annuus

As the central carbon uptake pathway in photosynthetic cells, the Calvin-Benson cycle is among the most important biochemical cycles for life on Earth. Recently, anaplerotic carbon flux (through the chloroplast-localised oxidative branch of the pentose phosphate pathway) into this cycle was proposed. Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO2 concentrations, Ca. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations. We report deuterium fractionation signals at starch glucose H1 and H2. Below a response change point, these signals increase with decreasing Ca consistent with modelled fractionations by anaplerotic flux. Under normal growth conditions (Ca≥450 ppm corresponding to intercellular CO2 concentrations, Ci, ≥328 ppm), we estimate negligible anaplerotic flux. At Ca=180 ppm (Ci=140 ppm), we estimate that of the glucose 6-phosphate entering the starch biosynthesis pathway more than 11.5% is diverted into the anaplerotic pathway. In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin-Benson cycle in vivo. We propose the flux may help to (i) maintain high levels of ribulose 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength and (ii) counteract oxidative stress.

Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica

ABSTRACT Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and β-methylmalyl-CoA lyase (haloarchaeal β-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave β-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a β-methylmalyl-CoA lyase reaction in vivo. The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to-be-discovered malyl-CoA thioesterase in haloarchaea. IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase by a Ca2+-dependent protein kinase suggests a link between Ca2+ signalling and anaplerotic pathway control in developing castor oil seeds

Inhibitory Ser451 phosphorylation of bacterial-type phosphoenolpyruvate carboxylase subunits of the Class-2 PEPC hetero-octameric complex of developing castor beans is catalysed by a Ca2+-dependent protein kinase. This suggests a relationship between Ca2+ signalling and the control of anaplerosis in developing oil seeds.

Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis

The glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid (TCA) cycle that allows growth on C2 compounds by bypassing the CO2-generating steps of the TCA cycle. The unique enzymes of this route are isocitrate lyase (ICL) and malate synthase (MS). ICL cleaves isocitrate to glyoxylate and succinate, and MS converts glyoxylate and acetyl-CoA to malate. The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The glyoxylate cycle occurs in Eukarya, Bacteria and Archaea. Recent studies of ICL- and MS-deficient strains as well as proteomic and transcriptional analyses show that these enzymes are often important in human, animal and plant pathogenesis. These studies have extended our understanding of the metabolic pathways essential for the survival of pathogens inside the host and provide a more complete picture of the physiology of pathogenic micro-organisms. Hopefully, the recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases.

13C isotopomer model for estimation of anaplerotic substrate oxidation via acetyl-CoA

A previous model using 13C nuclear magnetic resonance isotopomer analysis provided for direct measurement of the oxidation of 13C-enriched substrates in the tricarboxylic acid cycle and/or their entry via anaplerotic pathways. This model did not allow for recycling of labeled metabolites from tricarboxylic acid cycle intermediates into the acetyl-CoA pool. An extension of this model is now presented that incorporates carbon flow from oxaloacetate or malate to acetyl-CoA. This model was examined using propionate metabolism in the heart, in which previous observations indicated that all of the propionate consumed was oxidized to CO2 and water. Application of the new isotopomer model shows that 2 mM [3-13C]propionate entered the tricarboxylic acid cycle as succinyl-CoA (an anaplerotic pathway) at a rate equal to 52% of tricarboxylic acid cycle turnover and that all of this carbon entered the acetyl-CoA pool and was oxidized. This was verified using standard biochemical analysis; from the rate (mumol.min-1.g dry wt-1) of propionate uptake (4.0 +/- 0.7), the estimated oxygen consumption (24.8 +/- 5) matched that experimentally determined (24.4 +/- 3).