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

Abstract

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.

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

“…In contrast with many heterotrophic bacteria, Synechocystis citrate synthase (GltA) was shown only to catalyse generation of citrate, not its cleavage. Synechocystis GltA has a lower substrate affinity and turnover rate than the E. coli homologue, is not inhibited by ATP and NADH, but is inhibited by phosphoenolpyruvate [55].…”

“…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.…”

Biochemical characterisation of fumarase C from a unicellular cyanobacterium demonstrating its substrate affinity, altered by an amino acid substitution

“…Additional effectors of Sy FumC enzymatic activity were examined under conditions of substrate saturation. We selected 11 effectors which regulate the activities of enzymes in the TCA cycle and pyruvate metabolism in this cyanobacterium 37,41 . Among the 11 effectors tested, Co 2+ and Zn 2+ decreased Sy FumC activity towards fumarate and l- malate (Fig.…”

“…These results indicated that the regulation by metabolites and metals was not conserved among fumarases. Three TCA cycle enzymes ( Sy MDH, isocitrate dehydrogenase, and citrate synthase) in Synechocystis 6803 are regulated by divalent cations, particularly Mg 2+ 41,58,63 . Sy FumC was severely inhibited by Zn 2+ , not by Mg 2+ , indicating the different regulatory manner of enzymatic activities in the TCA cycle in this cyanobacterium.…”

AcnSP – A Novel Small Protein Regulator of Aconitase Activity in the Cyanobacterium Synechocystis sp. PCC 6803

“…It has also been shown that the relative flux into the TCA cycle of cyanobacteria changes in dependence from growth conditions, for example the availability of inorganic carbon or nitrogen ( Schwarz et al, 2013a ). Recently, it has been reported that citrate synthase of the Synechocystis 6803 TCA cycle is highly regulated and that the TCA cycle intermediate citrate regulates the activity of the oxidative pentose-phosphate (OPP) pathway ( Ito et al, 2019 ; Ito and Osanai, 2020 ).…”