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

Shoki Ito,Naoto Koyama,Takashi Osanai

doi:10.1038/s41598-019-42659-z

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 (SyCS) and compared its characteristics with other bacterial CSs. SyCS catalysed only the generation of citrate, and did not catalyse the cleavage of citrate. It is suggested that SyCS is not related to the reductive TCA cycle. The substrate affinity and turnover number of SyCS were lower than those of CSs from heterotrophic bacteria. SyCS was activated by MgCl2 and CaCl2, which inhibit various bacterial CSs. SyCS was not inhibited by ATP and NADH; which are typical feedback inhibitors of other bacterial CSs. SyCS was inhibited by phosphoenolpyruvate and activated by ADP, which has not been reported for CSs from heterotrophic bacteria. Thus, SyCS showed unique characteristics, particularly its sensitivity to effectors.

Highlights

Citrate synthase (CS, EC 2.3.3.1) catalyses the first reaction of the tricarboxylic acid (TCA) cycle: oxaloacetate + acetyl-CoA + H2O → citrate + CoA-SH
Characterising acetyl-CoA metabolism and the enzymes involved is crucial for understanding the basic science of cyanobacteria and its potential practical applications
For biochemical analysis of Synechocystis 6803 (SyCS), glutathione-S-transferase (GST)-tagged SyCS was purified from Escherichia coli by affinity chromatography (Fig. 2a)

Summary

Introduction

Citrate synthase (CS, EC 2.3.3.1) catalyses the first reaction of the tricarboxylic acid (TCA) cycle: oxaloacetate + acetyl-CoA + H2O → citrate + CoA-SH. In Synechocystis 6803, biochemical analyses of a phosphoenolpyruvate carboxylase (encoded by pps, sll0920) being the rate-limiting enzyme in succinate production[9,10] and d-lactate dehydrogenase (encoded by ddh, slr1556) catalysing the final reaction in d-lactate production have been performed (Fig. 1)[11,12] These reports identified the enzymatic properties and the critical amino acid residues regulating the enzymatic activities, and emphasised the importance of biochemical studies of this cyanobacterium[11,12]. Ethylene production from the TCA cycle intermediates in Synechocystis 6803 has been demonstrated (Fig. 1)[21,22] Despite these metabolic engineering applications, few studies have conducted biochemical analysis using acetyl-CoA as a substrate in cyanobacterium. We performed biochemical analyses of SyCS (encoded by gltA, sll0401) (Fig. 1) and compared its properties with those of other bacterial CSs

Methods

Results

Conclusion