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

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.

Citrate synthase (CS, EC 2.3.3.1) catalyses the first reaction of the tricarboxylic acid (TCA) cycle: oxaloacetate + acetyl-CoA + H 2 O → citrate + CoA-SH. Different bacterial CSs have been characterised, and studies have shown that the biochemical properties of CSs from Gram-positive bacteria and Gram-negative bacteria exhibit some differences. CSs from Gram-negative bacteria are inhibited by NADH, whereas CSs from Gram-positive bacteria are not 1,2 . However, the biochemical properties of cyanobacterial CSs have not been well-characterised.
Photoautotrophic cyanobacteria have been evaluated for use in the sustainable production of bioplastics and biofuels from carbon dioxide using light energy 3 . Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803) is a unicellular cyanobacterium that has been widely studied for use in metabolite production. Synechocystis 6803 exhibits suitable characteristics as a host for metabolite production such as rapid growth and natural transformation capability 4 . Synechocystis 6803 can be repeatedly genetically manipulated 5 . Various genetic toolsets have been developed for Synechocystis 6803 6 . This freshwater cyanobacterium can also grow in artificial seawater supplemented with nitrogen and phosphorus 7 . Under dark and anaerobic conditions, Synechocystis 6803 produces organic acids such as d-lactate and succinate, which are used in bioplastics production ( Fig. 1) 8 . 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 . For example, we have previously indicated that lower activity of d-lactate dehydrogenase (Ddh) from Synechocystis 6803 compared to other bacterial Ddh is one of the weak point of d-lactate production using Synechocystis 6803 12 . We have indicated that Ddh from Synechocystis 6803 also catalyses the reaction from oxaloacetate to malate and an amino acid substitution alters the substrate specificity of Ddh 12 .
Characterising acetyl-CoA metabolism and the enzymes involved is crucial for understanding the basic science of cyanobacteria and its potential practical applications. The intracellular acetyl-CoA concentration of Synechocystis 6803 is much higher compared to those of other cyanobacteria 13 . The production of

Results
Purification and kinetic analyses of SyCS. For biochemical analysis of SyCS, glutathione-S-transferase (GST)-tagged SyCS was purified from Escherichia coli by affinity chromatography (Fig. 2a). The optimal conditions at which SyCS showed maximal activity were 37 °C and pH 7.5 (Fig. 2b,c). Therefore, subsequent analysis of SyCS was conducted under these conditions. To calculate the kinetic parameters of SyCS, saturation curves of SyCS for both substrates were drawn (Fig. 3). The observed K m value of SyCS for oxaloacetate was 91 ± 11 μM and the k cat value for oxaloacetate was 2.76 ± 0.26 s −1 ( Table 1). The k cat /K m value of SyCS for oxaloacetate was 30.50 ± 3.17 s −1 mM −1 (Table 1). Similarly, for acetyl-CoA, the K m value was 220 ± 77 μM and the k cat value was 2.51 ± 0.24 s −1 ( Table 1). The k cat /K m value of SyCS for acetyl-CoA was 12.07 ± 2.95 s −1 mM −1 (Table 1). SyCS showed no catalytic activity for the backward reaction, generating acetyl-CoA and oxaloacetate from citrate and CoA.
Effects of ions on SyCS activity. The effects of monovalent and divalent salts on SyCS activity were examined. SyCS activity was altered by monovalent and divalent salts, which was affected by the concentration of substrates ( Fig. 4). At a saturated concentration of both substrates in the absence of effectors, SyCS activity increased to 188% in the presence of 100 mM KCl, 205% in the presence of 100 mM NaCl, 391% in the presence of 100 mM MgCl 2 , and 251% in the presence of 100 mM CaCl 2 , while enzyme activity was decreased to 48% in the presence of 100 mM MnCl 2 (Fig. 4). At the half-saturated concentration of both substrates in the absence of effectors, SyCS activity increased to 345% in the presence of 100 mM KCl, 336% in the presence of 100 mM NaCl, 1,463% in the presence of 100 mM MgCl 2 , and 1,050% in the presence of 100 mM CaCl 2 , while enzyme activity was decreased to 37% in the presence of 100 mM MnCl 2 (Fig. 4).
The activation of SyCS by monovalent and divalent salts was affected by the pH (Fig. 5). In KCl, SyCS activation was concentration-dependent at both pH 7.0 and pH 8.5 (Fig. 5a). The activity of SyCS increased linearly with increasing concentrations of NaCl at pH 7.0 ( Fig. 5b). At pH 8.5, SyCS activity did not increase up to 50 mM NaCl; however, it increased by 2.4-fold in the presence of 100 mM NaCl (Fig. 5b). The activation of SyCS by 100 mM monovalent salts at pH 8.5 was greater than that at pH 7.0 ( Fig. 5a,b). The activation of SyCS peaked at 20 mM MgCl 2 at both pH 7.0 and pH 8.5 (Fig. 5c). The activation of SyCS peaked at 20 mM CaCl 2 at pH 8.5 and at 50 mM CaCl 2 at pH 7.0 (Fig. 5d). In contrast to monovalent salts, the activation of SyCS by 100 mM divalent salts at pH 7.0 was greater than that at pH 8.5 (Fig. 5c,d).

Regulation of SyCS activity by metabolites.
SyCS activity was also altered by different metabolites (Fig. 6). In the presence of 5 mM phosphoenolpyruvate (PEP), citrate (CIT), and 2-oxoglutarate (2OG), SyCS activity markedly decreased to 14%, 1%, and 2%, respectively (Fig. 6). Although PEP is a metabolite in www.nature.com/scientificreports www.nature.com/scientificreports/ glycolysis, it inhibited SyCS activity (Fig. 6). To examine the changes in the kinetic parameters of SyCS, saturation curves of SyCS for both substrates in the presence of 3 mM PEP were drawn (Fig. 3). In the presence of 3 mM PEP, the K m value of SyCS for oxaloacetate was 108 ± 4 μM, the k cat value of SyCS for oxaloacetate decreased to 1.53 ± 0.06 s −1 , and the k cat /K m value of SyCS for oxaloacetate decreased to 14.19 ± 0.99 s −1 mM −1 (Table 1).  www.nature.com/scientificreports www.nature.com/scientificreports/ In the presence of 3 mM PEP, the K m value of SyCS for acetyl-CoA was 194 ± 36 μM, the k cat value of SyCS for acetyl-CoA decreased to 1.42 ± 0.18 s −1 , and the k cat /K m value of SyCS for acetyl-CoA was 7.39 ± 0.70 s −1 mM −1 ( Table 1). In contrast, SyCS activity significantly increased to 369% in the presence of 5 mM ADP (Fig. 6). To examine changes in the kinetic parameters of SyCS, the saturation curves of SyCS for both substrates in the presence of 5 mM ADP were drawn (Fig. 3). In the presence of 5 mM ADP, the K m value of SyCS for oxaloacetate was 73 ± 11 μM, the k cat value of SyCS for oxaloacetate was 3.35 ± 0.54 s −1 , and the k cat /K m value of SyCS for oxaloacetate increased to 45.65 ± 0.84 s −1 mM −1 (Table 1). In the presence of 5 mM ADP, the K m value of SyCS for acetyl-CoA was 153 ± 24 μM, the k cat value of SyCS for acetyl-CoA was 2.99 ± 0.12 s −1 , and the k cat /K m value of SyCS for acetyl-CoA increased to 19.90 ± 2.82 s −1 mM −1 (Table 1). Compounds related to the TCA cycle and amino acid metabolism [pyruvate (PYR), succinate (SUC), fumarate (FUM), l-malate (MAL), and l-aspartate (ASP)] and nucleotides (ATP, AMP, NADH, NAD + , NADPH, and NADP + ) did not significantly alter SyCS activity (Fig. 6).

Discussion
We purified and biochemically characterised CS from the cyanobacterium Synechocystis sp. PCC 6803 in this study. The k cat value of SyCS was lower than those of reported CSs from heterotrophic bacteria (8-263 s −1 ) 23-26 ( Table 1). The K m value of SyCS for acetyl-CoA was similar to that of CS from Arthrobacter strain DS2-3R (200 μM) 26 but higher than those of reported CSs from heterotrophic bacteria (14-200 μM) [23][24][25][26][27][28][29][30] (Table 1). Similarly, the K m value of SyCS for oxaloacetate was higher than those of CSs from heterotrophic bacteria (4-20 μM) [23][24][25][26][27][28][29][30] (Table 1). These observations indicate that SyCS 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 Table 1. Kinetic parameters of SyCS for oxaloacetate and acetyl-CoA. The experiment was conducted using 50 pmol of SyCS, and enzymatic activity was measured under optimal conditions (37 °C and pH 7.5). The concentrations of oxaloacetate or acetyl-CoA were 1 and 0.5 mM, respectively. The data represent the mean ± SD from three independent experiments. The asterisks indicated significant differences between the absence and presence of the effector (Student's t test; *P < 0.05). www.nature.com/scientificreports www.nature.com/scientificreports/ the observed low carbon flux through the oxidative TCA cycle in Synechocystis 6803. Additionally, CSs from Thermosulfidibacter takaii ABI70S6T 37 and Desulfurella acetivorans 38 catalyse the backward reaction, the cleavage of citrate. SyCS did not catalyse the backward reaction. This result suggests that SyCS is not involved in the reductive TCA cycle (Fig. 1).   (Figs 4-6). Monovalent salts activate numerous bacterial CSs 2 , while divalent salts inhibit many bacterial CSs 39,40 . In contrast to other bacterial CSs, SyCS was markedly activated by the divalent salts MgCl 2 and CaCl 2 rather than monovalent salts (Fig. 4). Among divalent salts, MnCl 2 inhibited SyCS (Fig. 4). In CS from E. coli, a higher pH leads to greater activation by 0.1 M KCl 39 . In SyCS, a higher pH led to greater activation by 0.1 M KCl and NaCl (Fig. 5a,b). In contrast, a lower pH led to greater activation by 0.1 M MgCl 2 and CaCl 2 (Fig. 5c,d). In monovalent and divalent salts, MgCl 2 was the strongest activator of SyCS (Figs 4 and 5). In the stroma in spinach chloroplasts, free Mg 2+ concentration is affected by light-dark transition 41 . It is expected that free Mg 2+ concentration in Synechocystis 6803 is similarly affected by light-dark transition 42 . Therefore, SyCS activity may also be affected by light-dark transition. NADH is a feedback inhibitor of CSs in most Gram-negative bacteria 1 . However, SyCS was not inhibited by NADH (Fig. 6). Phylogenetic analysis revealed that SyCS belongs to a cyanobacterial clade being different from clades of Gram-positive bacteria and Gram-negative bacteria (Fig. 7). Unlike heterotrophic bacteria, cyanobacteria do not possess 2-oxoglutarate dehydrogenase in the TCA cycle, and Synechocystis 6803 is rerouted to produce succinate by two alternate pathways ( Fig. 1) 43,44 . Isocitrate dehydrogenase from Synechocystis 6803 (encoded by icd, slr1289) ( Fig. 1) is specific to NADP + and generates NADPH but not NADH 45 . 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 SyCS. CIT and 2OG are feedback inhibitors of SyCS and several bacterial CSs 1,2,46 (Fig. 6). We demonstrated that PEP is a unique inhibitor of SyCS (Fig. 6). In the presence of PEP, the K m values of SyCS for both substrates were unchanged and the k cat values of SyCS for both substrates were decreased (Table 1). These results indicate that PEP is a non-competitive inhibitor of SyCS. Phosphoenolpyruvate carboxylase from Synechocystis 6803 (encoded by pps, sll0920) catalyses the carboxylation of PEP to generate oxaloacetate, a substrate of SyCS (encoded by gltA, sll0401) and malate dehydrogenase (encoded by citH, sll0891) ( Fig. 1) 11 . The reaction catalysed by phosphoenolpyruvate carboxylase (encoded by pps, sll0920) ( Fig. 1) is the rate-limiting step in the production of succinate via the reductive TCA cycle in Synechocystis 6803 9,10 . PEP has been suggested to regulate both the oxidative and reductive TCA cycle in Synechocystis 6803. The intracellular concentrations of CIT and PEP are much higher than that of 2OG (CIT: 7.3 times, PEP: 8.5 times) 13 . In inhibitors of metabolites, CIT and PEP may have large inhibitory effects on SyCS under in vivo conditions. While ATP did not alter SyCS activity (Fig. 6), it acts as a feedback inhibitor for other CSs because of its structural similarity with acetyl-CoA 1,2 . In terms of activators, AMP upregulates the activity of CSs from Azotobacter vinelandii 47 and Streptomyces diastaticus No. 7 strain M1033 24 . However, SyCS was activated by ADP rather than AMP (Fig. 6). ADP enhanced the catalytic efficiency (k cat /K m ) of SyCS (Table 1). Generally, ADP inhibits CS for the same reason as ATP 1,2 . Our study indicates that ADP is a unique activator of SyCS. Synechocystis 6803 has more ADP than ATP in the cells 13 in contrast to E. coli 48 whose CS is inhibited by ATP and hardly affected by ADP 49 . This may be a reason that SyCS is affected by not ATP but ADP.
Taken together, we found that the characteristics of SyCS significantly differ from those of other bacterial CSs. www.nature.com/scientificreports www.nature.com/scientificreports/ Methods Construction of the recombinant SyCS expression vector. The region containing gltA (sll0401, encoding SyCS) in the Synechocystis 6803 genome was commercially synthesized by Eurofin Genomics Japan (Tokyo, Japan) and cloned into the BamHI-XhoI site of the pGEX5X-1 vector (GE Healthcare, Little Chalfont, UK).

Affinity purification of recombinant proteins. The expression vector was transformed into E. coli
DH5α cells (TakaraBio, Shiga, Japan). A 2-L culture of transformed DH5α cells in LB media was prepared by shaking at 125 rpm at 30 °C overnight. Expression of the recombinant proteins was induced by 0.01 mM isopropyl β-d-1-thiogalactopyranoside (Wako Chemicals, Osaka, Japan). Affinity purification of the recombinant proteins was performed as described previously 12 . The DH5α cells were lysed by sonication (VC-750, EYELA, Tokyo, Japan) for 200 s at 20% intensity. The supernatant was collected after centrifugation (5,800 × g for 2 min at 4 °C) and 560 μL of Glutathione-Sepharose 4B resin (GE Healthcare) was added to the supernatant. The mixture was incubated on ice for 30 min with constant shaking. After 30 min, 1 mM ATP and 1 mM MgSO 4 ·7H 2 O were added to the mixture and shaken for 30 min at 37 °C. After centrifugation (5,800 × g for 2 min at 4 °C) to remove the supernatant, the resin was re-suspended in 700 μL of PBS-T (1.37 M NaCl, 27 mM KCl, 81 mM Na 2 HPO 4 ·12H 2 O, 14.7 mM KH 2 PO 4 , and 0.05% Tween-20) with 1 mM ATP/1 mM MgSO 4 ·7H 2 O. The resin was washed 10 times using PBS-T, and the recombinant proteins were eluted with 700 μL of GST elution buffer (50 mM Tris-HCl, pH 8.0, and 10 mM reduced glutathione) four times. The eluted protein fractions were concentrated using a VivaSpin 500 MWCO 50000 device (Sartorius, Göttingen, Germany). The protein concentration was measured using a Pierce BCA Protein Assay Kit (Rockford, IL, USA). To evaluate protein purity, SDS-PAGE followed by staining with InstantBlue (Expedion Protein Solutions, San Diego, CA, USA) was performed.
Enzyme assays. The enzyme activity of SyCS for the forward reaction, generating citrate and CoA from acetyl-CoA and oxaloacetate, was assessed as described previously 50 . Enzyme activity was calculated by measuring the change in A 412 using a Hitachi U-3310 spectrophotometer (Tokyo, Japan). The purified SyCS proteins were added to 1 mL assay solution [50 mM Tris-HCl containing various concentrations of oxaloacetate, various concentrations of acetyl-CoA, and 0.2 mM DTNB]. The K m and V max values of SyCS were calculated using a Lineweaver-Burk double reciprocal plot. The k cat values of SyCS were determined from the V max values of SyCS. The enzyme activity of SyCS for the backward reaction, generating acetyl-CoA and oxaloacetate from citrate and CoA, was assessed as described previously 38 by measuring the change in A 365 using a Hitachi U-3310 spectrophotometer. The purified SyCS proteins were added to 1 mL assay solution [50 mM Tris-HCl containing 1 mM CoA, 1 mM citrate, 5 mM MgCl 2 , 0.5 mM NADH, and 20 U malate dehydrogenase (Oriental Yeast, Tokyo, Japan)].