Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

Takeya, Masahiro; Ito, Shoki; Sukigara, Haruna; Osanai, Takashi

doi:10.3389/fpls.2018.00947

Cited by 27 publications

(35 citation statements)

References 46 publications

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“…Synechocystis also encodes a succinyl-CoA synthetase complex (SucC/SucD), which probably catalyses the reversible conversion of succinate into succinyl-CoA in cyanobacteria [54], required for biosynthesis of methionine and lysine. Several recent papers have investigated the enzymatic properties of TCA enzymes conserved between cyanobacteria and heterotrophic bacteria [55][56][57]. In contrast with many heterotrophic bacteria, Synechocystis citrate synthase (GltA) was shown only to catalyse generation of citrate, not its cleavage.…”

Section: The Tricarboxylic Acid Cyclementioning

confidence: 99%

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

2020

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Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterizing cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialized compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterized. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and haem, is incomplete. We discuss tools that may aid our understanding of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterization of individual proteins.

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Section: The Tricarboxylic Acid Cyclementioning

confidence: 99%

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

2020

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“…These observations indicate that Sy CS is an inefficient enzyme, similar to malate dehydrogenase from Synechocystis 6803 (encoded by citH , sll0891), which is involved in the oxidative TCA cycle (Fig. 1) 31 . In Synechocystis 6803, carbon flux through the TCA cycle is low under heterotrophic, photoheterotrophic, photomixotrophic, and photoautotrophic conditions in contrast to other metabolic pathways such as the glycolysis and oxidative pentose phosphate pathways 22,32–36 .…”

Section: Discussionmentioning

confidence: 94%

“…Additionally, malate dehydrogenase from Synechocystis 6803 (encoded by citH , sll0891) (Fig. 1) specifically catalyses the reductive reaction generating NAD + but not NADH 31 . These previous studies explain why NADH does not function as a feedback inhibitor of Sy CS.…”

Section: Discussionmentioning

confidence: 99%

Citrate synthase from Synechocystis is a distinct class of bacterial citrate synthase

Ito

Koyama

Osanai

2019

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Citrate synthase (CS, EC 2.3.3.1) catalyses the initial reaction of the tricarboxylic acid (TCA) cycle. Although CSs from heterotrophic bacteria have been extensively studied, cyanobacterial CSs are not well-understood. Cyanobacteria can produce various metabolites from carbon dioxide. Synechocystis sp. PCC 6803 ( Synechocystis 6803) is a cyanobacterium used to synthesize metabolites through metabolic engineering techniques. The production of acetyl-CoA-derived metabolites in Synechocystis 6803 has been widely examined. However, the biochemical mechanisms of reactions involving acetyl-CoA in Synechocystis 6803 are poorly understood. We characterised the CS from Synechocystis 6803 ( Sy CS) and compared its characteristics with other bacterial CSs. Sy CS catalysed only the generation of citrate, and did not catalyse the cleavage of citrate. It is suggested that Sy CS is not related to the reductive TCA cycle. The substrate affinity and turnover number of Sy CS were lower than those of CSs from heterotrophic bacteria. Sy CS was activated by MgCl 2 and CaCl 2 , which inhibit various bacterial CSs. Sy CS was not inhibited by ATP and NADH; which are typical feedback inhibitors of other bacterial CSs. Sy CS was inhibited by phosphoenolpyruvate and activated by ADP, which has not been reported for CSs from heterotrophic bacteria. Thus, Sy CS showed unique characteristics, particularly its sensitivity to effectors.

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“…We have performed reconstitution of oxaloacetate metabolism in the cyanobacterial TCA cycle in vitro and examined the effects of factors such as pH and metabolites on oxaloacetate metabolism and the function of two L-malate dehydrogenase isozymes in oxaloacetate metabolism. Although the optimum temperatures for SyMDH (45°C) and SyCS (37°C) are different (Takeya et al, 2018;Ito et al, 2019), temperature did not have a remarkable effect on the L-malate/citrate ratio ( Figure S1). The optimum temperature of SyMDH (45°C) is higher than the predicted intracellular temperature of Synechocystis 6803 (below 40°C) (Takeya et al, 2018).…”

Section: Discussionmentioning

confidence: 96%

“…All P-values calculated by Student's t-test are listed in Table S1. The substrate concentrations were fixed at the K m (the concentration at 50% V max ) of the optimum conditions for SyMDH (oxaloacetate 0.012 mM, NADH 0.03 mM) (Takeya et al, 2018). For this assay, 150 pmol of SyMDH was used.…”

Section: Sensitivity To Mgcl 2 Is Different For Symdh and Sycsmentioning

confidence: 99%

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate

et al. 2020

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The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg 2+ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two L-malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.

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Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle

Cited by 27 publications

References 46 publications

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

Citrate synthase from Synechocystis is a distinct class of bacterial citrate synthase

Reconstitution of oxaloacetate metabolism in the tricarboxylic acid cycle in Synechocystis sp. PCC 6803: discovery of important factors that directly affect the conversion of oxaloacetate

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