Introduction

Cyanobacteria are bacteria that can perform oxygenic photosynthesis and produce a variety of metabolites from carbon dioxide. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a well-studied model cyanobacterium, as its genome has been sequenced1 and it can be easily transformed and has the ability to multiply rapidly.

The tricarboxylic acid (TCA) cycle is one of the most important bacterial metabolic pathways. The oxidative TCA cycle produces 2-oxoglutarate (2-OG), a precursor for amino acid production, from oxaloacetate, citrate, and isocitrate2,3. Aconitase (EC 4.2.1.3) is the enzyme that catalyses the second reaction in the TCA cycle, i.e., it reversibly isomerises citrate and isocitrate via cis-aconitate4. This enzyme is encoded by the acnB gene and contains a [4Fe-4S] cluster. Bacterial aconitase is a bifunctional protein, and it binds to mRNA when the Fe-S cluster is disrupted by lack of iron and oxidative stress, thereby regulating gene expression5,6. In the cells of Synechocystis 6803, Fe-S clusters are generated by Suf proteins and inserted into apo-proteins7,8. There are two genetically distinct aconitases in bacteria. Escherichia coli possesses two aconitases, namely, aconitase A (AcnA) and aconitase B (AcnB); AcnB is unstable under in vitro conditions9. The amino acid sequences of the two enzymes are approximately 17% identical10. In E. coli, AcnB is the major enzyme of the TCA cycle and is synthesised during the exponential growth phase, whereas AcnA is expressed during the stationary phase under conditions of iron deficiency and oxidative stress11,12.

The genes involved in the oxidative TCA cycle in cyanobacteria are essential13, and the cyanobacterial TCA cycle was thought to be incomplete, as cyanobacteria lack 2-oxoglutarate dehydrogenase. Synechocystis 6803 can convert 2-OG to succinate by two alternative pathways. The first pathway involves two enzymes, namely, 2-OG decarboxylase and succinic semialdehyde dehydrogenase14,15, and the second pathway is the γ-aminobutyric acid shunt pathway16. Intracellular citrate levels in Synechocystis 6803 are 10-fold higher than malate, fumarate, succinate, and 2-OG levels and 1.5-fold higher than the isocitrate levels17. These results suggest that citrate functions as a pool of carbon source in this cyanobacterium. Citrate also plays a key role in the regulation of sugar metabolism in Synechocystis 6803 because it specifically inhibits the enzymes of the oxidative pentose phosphate pathway, namely, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase18. Additionally, the expression and abundance of Synechocystis 6803 aconitase (SyAcnB) vary according to culture conditions. SyAcnB abundance increases by 3.8-fold following 48 h of nitrogen depletion, compared to that under photoautotrophic conditions19. Furthermore, the acnB transcript levels increase by more than 2-fold of the original level after 2 h of nitrogen depletion in Synechocystis 680320. These results indicate that it is important for Synechocystis 6803 to regulate the citrate level and its related enzyme aconitase to adapt to environmental changes.

However, limited information is available about the biochemical properties of aconitase in bacteria containing only AcnB. The Vmax and Km values of SyAcnB for cis-aconitate have been determined21, but the biochemical characteristics of SyAcnB using citrate and isocitrate as substrates have not been investigated. In this study, we determined the optimal conditions, kinetic parameters, and the influence of other TCA metabolites on SyAcnB using citrate and isocitrate as substrates. Overall, our biochemical analyses elucidated the metabolic flow of citrate in Synechocystis 6803.

Results

Purification and reactivation conditions of SyAcnB

To determine whether aconitase is the only enzyme in Synechocystis 6803 that uses citrate as a substrate, BLAST search was performed. Synechocystis 6803 did not possess genes encoding ATP-citrate lyase (ACL) and citryl-CoA synthase/citryl-CoA lyase (CCS/CCL) which cleave citrate (Table 1). The results of the BLAST search showed that Synechocystis 6803 possesses only AcnB (Table S1).

Table 1 BLAST search results for ATP-citrate lyase, citryl-CoA synthetase, and citryl-CoA lyase.
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We expressed the GST-tagged SyAcnB in E. coli DH5α and purified it using affinity chromatography (Fig. 1a). No SyAcnB activity was observed in the apoenzyme state, (without reactivation). The activity of SyAcnB with citrate as the substrate was 76% of its maximum activity at 1 min after the addition of the reagents; the activity peaked at 20 min and then gradually decreased (Fig. 1b). The activity of SyAcnB for citrate increased depending on the concentration of the reducing agent DTT (1–5 mM) added for enzyme reactivation (Fig. 1c). Hereafter, the reactivation of SyAcnB was carried out with 5 mM DTT for 20 min, similar to a previous study22. Na2S and (NH4)2Fe(SO4)2·6H2O were added to the mixture after the addition of DTT.

Figure 1
figure 1

Determination of reactivation conditions for Synechocystis sp. PCC 6803 aconitase B (SyAcnB). (a) Affinity purification results for GST-tagged SyAcnB. The purified protein was electrophoresed via 8% SDS-PAGE, and the gel was stained using Instant Blue reagent. (b) Effect of reactivation time on SyAcnB activity. The experiment was performed using 50 pmol of SyAcnB and 20 mM trisodium citrate dihydrate in Tris–HCl buffer pH 8.0 at 45 °C. Mean ± SD values were calculated from three independent experiments. (c) Effect of DTT concentration on SyAcnB activity. The experiment was performed using 50 pmol of SyAcnB and 20 mM trisodium citrate dihydrate in Tris–HCl buffer pH 7.7 at 45 °C. Mean ± SD values were calculated from three independent experiments. GST, glutathione-S-transferase; SDS-PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; DTT, dl-dithiothreitol; SD, standard deviation.

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Kinetic parameters of SyAcnB

The activity of SyAcnB for citrate was the highest at pH 7.7 and temperature 45–55 °C (Fig. 2a), and that for isocitrate as the substrate was the highest at pH 8.0 and a temperature of 53 °C (Fig. 2b). Thereafter, the activities of SyAcnB for citrate were measured at pH 7.7 and 45 °C and for isocitrate at pH 8.0 and 53 °C except where indicated.

Figure 2
figure 2

Optimal pH and temperature for SyAcnB. (a) Effect of pH on SyAcnB activity. The experiment was performed using 50 pmol of SyAcnB. The red circles and triangles represent the specific activity for citrate at 45 °C. The blue circles and triangles represent the specific activity for isocitrate at 53 °C. Mean ± SD values were calculated from three independent experiments. (b) Effect of temperature on SyAcnB activity. This was measured in Tris–HCl buffer pH 8.0. Mean ± SD values were calculated from three independent experiments. SyAcnB, Synechocystis sp. PCC 6803 aconitase B; SD, standard deviation.

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The kinetic parameters of SyAcnB, using citrate and isocitrate as the substrates, were estimated from the saturation curves (Fig. 3a,b). The Vmax, kcat, and kcat/Km values of the activity of SyAcnB for citrate were 4.58 ± 0.07 unit/mg, 9.12 ± 0.14 s−1, and 8.11 ± 0.23 s−1 mM−1, respectively (Table 2). The Vmax, kcat, and kcat/Km values of the activity of SyAcnB for isocitrate were 8.36 ± 0.17 unit/mg, 16.67 ± 0.34 s−1, and 10.88 ± 0.93 s−1 mM−1, respectively (Table 2). The Km values of SyAcnB for citrate and isocitrate were 1.13 ± 0.04 and 1.54 ± 0.17 mM, respectively (Table 3).

Figure 3
figure 3

Saturation curves displaying the activities of SyAcnB. The experiment was performed using (a) trisodium citrate dihydrate in Tris–HCl buffer pH 7.7 at 45 °C and (b) dl-isocitrate trisodium salt hydrate in Tris–HCl buffer pH 8.0 at 53 °C. Mean ± SD values were calculated from three independent experiments. SyAcnB, Synechocystis sp. PCC 6803 aconitase B; SD, standard deviation.

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Table 2 Kinetic parameters of SyAcnB.
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Table 3 List of Km values with aconitase from various organisms.
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Kinetic parameters were calculated under optimal conditions for both substrates. Therefore, we calculated the parameters by unifying the measurement conditions and plotting a substrate saturation curve at 30 °C, which is the optimal temperature for the growth of Synechocystis 680323 (Fig. 4a,b). In the presence of Tris–HCl (pH 7.0) at 30 °C, the Vmax and kcat/Km values of SyAcnB for citrate were 3.65 ± 0.19 unit/mg and 10.68 ± 0.76 s−1 mM−1, respectively (Table 2). The Vmax and kcat/Km values for isocitrate were 3.19 ± 0.18 unit/mg and 30.17 ± 1.51 s−1 mM−1 respectively and 0.87- and 2.8-fold higher than those for citrate, respectively (Table 2). In the presence of Tris–HCl (pH 8.0) at 30 °C, the Vmax and kcat/Km values of SyAcnB for citrate were 3.51 ± 0.21 unit/mg and 8.79 ± 0.27 s−1 mM−1, respectively (Table 2). The Vmax and kcat/Km values for isocitrate were 4.52 ± 0.31 unit/mg and 19.13 ± 1.12 s−1 mM−1 respectively and 1.3- and 2.2-fold higher than those for citrate, respectively (Table 2). Finally, in the presence of Tris–HCl (pH 9.0) at 30 °C, the Vmax and kcat/Km values of SyAcnB for citrate were 2.22 ± 0.09 unit/mg and 1.95 ± 0.22 s−1 mM−1, respectively (Table 2). The Vmax and kcat/Km values for isocitrate were 2.61 ± 0.11 unit/mg and 3.18 ± 0.47 s−1 mM−1 respectively and 1.2- and 1.6-fold higher than those for citrate, respectively (Table 2). The Km values of the activity of SyAcnB for citrate at 30 °C were 0.68 ± 0.02 mM, 0.80 ± 0.03 mM, and 2.28 ± 0.16 mM at pH 7.0, 8.0, and 9.0, respectively, and the values for citrate were 3.2-, 1.7-, and 1.4-fold higher than those calculated for isocitrate at pH 7.0, 8.0, and 9.0, respectively (Table 3). Since there were some points where the correlation coefficient (R2 value) was low at 30 °C, the same measurement was performed at 45 °C (Fig. 4c,d). In the presence of Tris–HCl (pH 9.0) at 45 °C, the Vmax, Km and kcat/Km values of SyAcnB for citrate were 2.88 ± 0.17 unit/mg, 1.58 ± 0.27 mM and 3.69 ± 0.61 s−1 mM−1, respectively, and the Vmax, Km and kcat/Km values for isocitrate were 4.64 ± 0.60 unit/mg, 3.79 ± 1.18 mM and 2.54 ± 0.50 s−1 mM−1 respectively (Table 4). All Km values are summarised in Table S2. The results of adding the peptide AcnSP (aconitase small protein) showed that the Vmax, Km, and kcat/Km values of SyAcnB for citrate were 4.29 ± 0.08 unit/mg, 0.74 ± 0.09 mM, and 11.65 ± 1.24 s−1 mM−1, respectively, and the Vmax, Km, and kcat/Km values of SyAcnB for isocitrate were 6.59 ± 0.08 unit/mg, 0.91 ± 0.05 mM, and 14.50 ± 0.72 s−1 mM−1, respectively (Fig. S1). For both citrate and isocitrate, the addition of peptide AcnSP decreased the Vmax and Km values and increased the kcat/Km value.

Figure 4
figure 4

Saturation curves displaying the activities of SyAcnB at each pH. The experiment was performed using (a) trisodium citrate dihydrate and (b) dl-isocitrate trisodium salt hydrate in Tris–HCl buffer pH 7.0, 8.0, and 9.0 at 30 °C. The experiment was repeated using (c) trisodium citrate dihydrate and (d) dl-isocitrate trisodium salt hydrate in Tris–HCl buffer pH 7.0, 8.0, and 9.0 at 45 °C. Mean ± SD values were calculated from three independent experiments. SyAcnB, Synechocystis sp. PCC 6803 aconitase B; SD, standard deviation.

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Table 4 Kinetic parameters of SyAcnB.
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The activity of SyAcnB in the presence of other TCA metabolites and cations

We examined the effects of various metabolites on SyAcnB activity. The concentrations of the substrates used were the Km values determined for each substrate. In the presence of 5 mM pyruvate, 2-OG, and l-aspartate, the activity of SyAcnB for citrate decreased to 69%, 72%, and 84% of that of the control, respectively (Fig. 5a). Additionally, in the presence of 5 mM pyruvate, 2-OG, l-glutamine, l-glutamate, and l-aspartate, the activity of SyAcnB for isocitrate decreased to 78%, 74%, 81%, 85%, and 89% of that of the control, respectively (Fig. 5b). The kinetic parameters of SyAcnB in the absence (Table 2, 3) and the presence of 2-OG under optimal conditions (Fig. S2, S3) were compared. When citrate was used as a substrate, the addition of 1 mM 2-OG did not change the Vmax, Km, and kcat/Km values (Fig. S2a), but the addition of 5 mM 2-OG increased the Km value and decreased the kcat/Km value (Fig. S3a). When isocitrate was used as a substrate, the addition of 1 mM 2-OG decreased the Vmax and Km values and increased kcat/Km values (Fig. S2b), but the addition of 5 mM 2-OG decreased only the Vmax value (Fig. S3b). As the parameters changed differently depending on the 2-OG concentration, the effect of 2-OG was studied by adding 0.44 mM 2-OG, similar to intracellular concentrations24, at 30 °C under three different pH conditions. The substrate concentrations were set to the Km values listed in Table 2, respectively. There was no effect on the SyAcnB activity irrespective of 2-OG presence at three pH conditions (Fig. S4).

Figure 5
figure 5

Effect of other TCA metabolites on the activity of SyAcnB. The effect was analysed using (a) 1.13 mM trisodium citrate dihydrate and Tris–HCl buffer pH 7.7 at 45 °C and (b) 1.54 mM dl-isocitrate trisodium salt hydrate and Tris–HCl buffer pH 8.0 at 53 °C. Mean ± SD values were calculated from three independent experiments. The asterisks indicate significant differences compared to the SyAcnB activities at the control condition (Student’s t-test; *P < 0.05, **P < 0.005). SyAcnB, Synechocystis sp. PCC 6803 aconitase B; SD, standard deviation; NO, no enzymatic activity was detected; PEP, phosphoenolpyruvic acid monopotassium salt; Pyr, sodium pyruvate; G6P, glucose 6-phosphate; 2-OG, 2-oxoglutarate; Suc, disodium succinate; Mal, sodium l-malate; SSA, succinic semialdehyde; Gln, l-glutamine; Glu, l-glutamate; Asp, sodium l-aspartate monohydrate.

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Furthermore, we examined the effects of monovalent and divalent cations on SyAcnB activity. K+ had little effect on the activity of SyAcnB for citrate, whereas the activity decreased to 59% and 14% in the presence of 1 mM and 5 mM Ca2+, respectively, and 58% and 9% in the presence of 1 mM and 5 mM Mg2+, respectively (Fig. 6a). Unlike the results obtained for citrate, 5 mM Mg2+ decreased the activity of SyAcnB for isocitrate to 75%, and Ca2+ had little effect on the activity of SyAcnB for isocitrate (Fig. 6b). The activity of SyAcnB for citrate decreased to 6% and 35% in the presence of 1 mM Zn2+ and Mn2+, respectively, and 9% with 5 mM Zn2+ (Fig. 6a). Similarly, the activity of SyAcnB for isocitrate decreased to 6% and 23% in the presence of 1 mM Zn2+ and Mn2+, respectively, and 3% and 7% in the presence of 5 mM Zn2+ and Mn2+, respectively (Fig. 6b).

Figure 6
figure 6

Effect of various cations on the activity of SyAcnB. (a) The experiment was performed using (a) 1.13 mM trisodium citrate dihydrate in Tris–HCl buffer pH 7.7 at 45 °C and (b) 1.54 mM dl-isocitrate trisodium salt hydrate in Tris–HCl buffer pH 8.0 at 53 °C. The asterisks indicate significant differences compared to the SyAcnB activities at the control condition (Student’s t-test; *P < 0.05, **P < 0.005). ND, enzymatic activity not determined; K, KCl; Ca, CaCl2; Mg, MgCl2·6H2O; Zn, ZnSO4·7H2O; Mn, MnCl2·4H2O. (c) The activities were measured using 1.13 mM trisodium citrate dihydrate and Tris–HCl buffer pH 7.7 at 45 °C in the presence of (c) 1–5 mM Mg2+ or Ca2+, (d) 1 mM Mg2+, and (e) 1 mM Ca2+. Mean ± SD values were calculated from three independent experiments. SyAcnB, Synechocystis sp. PCC 6803 aconitase B; SD, standard deviation.

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We examined the effects of Mg2+ and Ca2+ on the kinetic parameters of the activity of SyAcnB for citrate. The inhibitory effects of Mg2+ and Ca2+ on the activity of SyAcnB for citrate were concentration-dependent (1–5 mM) (Fig. 6c). In the presence of 1 mM Mg2+, the Vmax of the activity of SyAcnB for citrate was 5.66 ± 0.20 unit/mg, and its Km value increased to 3.01 ± 0.04 mM, whereas its kcat/Km value decreased to 3.76 ± 0.12 s−1 mM−1 (Fig. 6d). Similarly, in the presence of 1 mM Ca2+, the Vmax of the activity of SyAcnB for citrate was 5.26 ± 0.49 unit/mg, and its Km value increased to 2.61 ± 0.28 mM, whereas its kcat/Km value decreased to 4.01 ± 0.10 s−1 mM−1 (Fig. 6e).

Discussion

In this study, we demonstrated the biochemical properties of aconitase, which preferentially catalyses the reaction from isocitrate to citrate, in the unicellular cyanobacterium Synechocystis 6803 using citrate and isocitrate as the substrates.

We investigated the reactivation conditions for in vitro enzymatic reaction by altering the DTT concentration and incubation time. Previous studies have suggested the requirement of varying concentrations of DTT, such as 5 mM22 or 1 mM25,26, for aconitase reactivation. We also revealed that the maximum activity of the enzyme varied with DTT concentration. Additionally, various incubation times have been suggested for aconitase reactivation, for example, 20 min at 25 °C26 and 30–120 min on ice22. We demonstrated that aconitase was reactivated immediately after the addition of the reagents, and its maximum activity gradually decreased after 20 min. AcnB from E. coli is reactivated faster than AcnA, but the enzyme is unstable10. Thus, a long reactivation period for SyAcnB may degrade the Fe-S cluster and reduce its activity.

The optimal pH required for Corynebacterium glutamicum aconitase (for citrate) is 7.5–7.826 and that for Mycobacterium tuberculosis aconitase (for isocitrate) is 8.027. These values are similar to the optimal pH values required for SyAcnB activity in the presence of citrate and isocitrate (Fig. 2a). The intracellular pH of Synechocystis 6803 in logarithmically growing cells has been reported to be approximately 7.5–7.7 under dark conditions28. This suggests that the optimal pH required for SyAcnB activity is suitable for the growth of Synechocystis 6803.

The optimal temperature required for SyAcnB activity was estimated to be 45–55 °C (Fig. 2b), which is higher than the optimal growth temperature (30 °C) required for Synechocystis 6803, as reported in a previous study23. The optimal temperature required for the maximum activity of aconitase from C. glutamicum and the thermophilic archaea Sulfolobus acidocaldarius has been reported to be approximately 50 °C and 75 °C, respectively26,29. Additionally, the optimal temperature required for the maximum activity of aconitase from C. glutamicum is higher than its optimal growth temperature (30 °C)30. As the optimal temperature required for aconitase activity is known only for a few microorganisms, it remains unknown whether the optimal temperature for aconitase activity is usually higher than that required for the growth of microorganisms, as in this case. However, this pattern has been observed in some enzymes of the TCA cycle in Synechocystis 6803, such as fumarase (SyFum) (30 °C)31, wherein the optimal temperature required for enzyme activity corresponds with the optimal growth temperature of the bacterium; on the other hand, the optimal temperature required for the activity of other enzymes, such as citrate synthase (CS) from Synechocystis 6803 (SyCS) (37 °C)32 and malate dehydrogenase (MDH) from Synechocystis 6803 (SyMDH) (45–50 °C)33, is higher than the optimal growth temperature of the bacterium. Enzymes are thermally denatured and inactivated at high temperatures; however, the reaction rate increases with the increase in temperature. Therefore, the optimal temperature required for the activity of some enzymes may be higher than the optimal growth temperature of the microorganisms.

The affinity of SyAcnB for citrate has been reported to be higher than that of aconitases from other microorganisms, namely, E. coli, S. acidocaldarius, and Salmonella enterica (Table 3)25,29,34. On the contrary, the affinity of SyAcnB for isocitrate has been reported to be lower than that of aconitase from other microorganisms such as E. coli, C. glutamicum, S. acidocaldarius, and S. enterica (Table 3)25,26,29,34. The Km value of the activity of aconitase from C. glutamicum for citrate was slightly lower than that for isocitrate, which is consistent with the results obtained for SyAcnB, whereas the Km values of the activity of aconitase from E. coli, S. acidocaldarius, S. enterica, Rattus norvegicus (mitochondrial), and Zea mays (mitochondrial) for citrate are higher than those reported for isocitrate (Table 3)25,26,29,34,35,36. The calculated values for Km (citrate)/Km (isocitrate) ratio are shown in Table 3; the ratio was estimated to be 0.73 at the optimum activity of SyAcnB, which is close to that for C. glutamicum aconitase (0.87). At pH 7.0, 8.0, and 9.0, the ratios were above 1 but were lower than those estimated for other organisms, except for C. glutamicum (Table 3). These results suggest that the aforementioned microorganisms tend to oxidise citrate to isocitrate. These values correspond with higher intracellular concentrations of citrate than isocitrate in Synechocystis 6803, which was estimated by the absolute quantification of metabolites17. The kcat/Km value of the activity of SyAcnB for isocitrate was slightly higher than that for citrate; this value is similar to that for aconitase from C. glutamicum (40.8 s−1 mM−1 for citrate and 52.4 s−1 mM−1 for isocitrate) and S. enterica (1.00 s−1 mM−1 for citrate and 1.22 s−1 mM−1 for isocitrate)26,34. Unlike heterotrophic bacteria, the TCA cycle flux in Synechocystis 6803 is always low under photoautotrophic, photomixotrophic, and heterotrophic conditions37,38,39,40, which may explain why the kcat/Km value of SyAcnB is lower than that of C. glutamicum aconitase.

The higher the pH, the lower the Km (citrate)/Km (isocitrate) ratio of SyAcnB (Table 3). The direction of the TCA cycle in Synechocystis 6803 is strongly affected by the pH, and the in vitro reconstruction of oxaloacetate metabolism displays a higher yield of citrate at higher pH41. At higher pH, higher concentrations of citrate, which is the substrate for SyAcnB, are formed, and the reaction is more likely to proceed in the oxidative direction at chemical equilibrium.

The TCA cycle in Synechocystis 6803 is characterised by the citrate accumulation at high levels in the cells, although 2-OG is generated through the oxidative TCA cycle from citrate under normal phototrophic growth conditions17. In Synechocystis 6803, isocitrate dehydrogenase (ICD) can catalyse isocitrate to form 2-OG. The Km value of the activity of ICD from Synechocystis 6803 (SyICD) for isocitrate was estimated to be 5.7 × 10–3–5.9 × 10–2 mM42. The Km values of the activity of SyAcnB for isocitrate were 3.6-fold higher than those of the activity of SyICD. Therefore, isocitrate is thought to be metabolised mainly by SyICD, rather than by SyAcnB, enabling the cells to produce 2-OG. The three lines of evidence, 1) the high level of citrate accumulation in Synechocystis 6803, 2) the lack of citrate-metabolising enzymes such as ACL and CCS/CCL43,44,45 (Table 1), and 3) the lack of SyCS activity degrading a citrate32, suggest that SyAcnB enhances the reaction in the direction of citrate to isocitrate to produce 2-OG. In this way, the properties of two enzymes, SyAcnB and SyICD, facilitate citrate accumulation and 2-OG generation at the same time. The peptide AcnSP affects the kinetic parameters of SyAcnB21 and we performed biochemical analysis using AcnSP (Fig. S1). In both cases, Vmax and Km values decreased as in previous studies using cis-aconitate as a substrate, suggesting that AcnSP does not a significant effect on the reaction direction but boosting the reaction between citrate and isocitrate catalysed by SyAcnB.

SyAcnB activities in both directions were inhibited by 2-OG in our study (Fig. 5); however, 2-OG has not been reported as an inhibitor of aconitase thus far. Therefore, we tested the effects of 2-OG in detail by comparing the kinetic parameters when 1 or 5 mM 2-OG was added (Fig. S2, S3) with those when it was not added (Table 2). When citrate was used as a substrate, 5 mM 2-OG acts as an inhibitor (Fig. S3a), but not at 1 mM 2-OG deduced from kcat/Km values of SyAcnB (Fig. S2a). Whereas when isocitrate was used as a substrate, 1 mM 2-OG acts as an activator (Fig. S2b), but not at 5 mM 2-OG deduced from kcat/Km values of SyAcnB (Fig. S3b). The addition of 0.44 mM 2-OG, the intracellular concentration in Synechocystis cells24, did not decrease SyAcnB activities (Fig. S4), and hence, 2-OG could play a role in the inhibition of SyAcnB activities when too many reactions of the oxidative reaction of the TCA cycle have proceeded. We also found that the activity of aconitase from Z. mays (mitochondrial) is inhibited by succinate and malate36, whereas that of SyAcnB was not inhibited by these organic acids.

The activity of SyAcnB for citrate was strongly inhibited by Mg2+ and Ca2+ ions (Fig. 6a). Mg2+ and Ca2+ increased the Km value. The kcat/Km values for citrate in the presence of 1 mM Mg2+ and Ca2+ were estimated to be 46% and 49% of the control, respectively. As per a previous report, the citrate/isocitrate concentration ratio for aconitase from rat heart was altered by Mg2+ and Ca2+ ions, and the equilibrium leaned towards citrate46. Comparing the effects of Mg2+ and Ca2+ on the activities of enzymes in the TCA cycle from Synechocystis 6803 revealed that the activity of SyCS increased to 1463% and 1050% of the control in the presence of 100 mM Mg2+ and Ca2+, respectively, and that the activity of SyMDH increased to 160% and 190% of the control in the presence of 1 mM and 10 mM Mg2+, respectively32,33. Additionally, SyICD requires Mg2+ or Mn2+ as a cofactor for its activity42. The concentration of free Mg2+ ions in the stroma of spinach chloroplasts varies between dark and light conditions47. Thus, depending on culture conditions, the concentration of free Mg2+ in Synechocystis 6803 cells may be altered48, which may affect the equilibrium of aconitase. Also, SyAcnB activities in both directions were strongly inhibited by Mn2+ and Zn2+ (Fig. 6a,b). The mitochondrial aconitase activity from rat AF5 cells decreased to 48% and 19% of the control in the presence of 2 mM and 5 mM Mn2+, respectively49. The activity of aconitase from rat prostate epithelial cells for citrate was inhibited by Zn2+, but this effect was not observed for isocitrate50. The activity of SyCS decreased to 37% of that in the control in the presence of 100 mM Mn2+32, and the activity of SyFum was inhibited by 10 mM Mn2+ when l-malate was used as a substrate31. Moreover, the activity of SyFum was strongly inhibited by 1 mM Zn2+31. Presently, the understanding of the physiological significance of metal ions in Synechocystis 6803 is limited.

In this study, we determined the biochemical properties of SyAcnB and demonstrated that citrate accumulation depends on the enzyme kinetics of SyAcnB. The consumption of isocitrate by SyICD to produce 2-OG overcomes the kinetic barrier of the SyAcnB enzyme. Currently, the study is limited to biochemical analysis; further genetic manipulation of SyAcnB might reveal its importance in citrate metabolism in cyanobacteria.

Methods

Construction of cloning vector for the expression of recombinant SyAcnB

The nucleotide sequence of acnB (slr0665), obtained from the sequenced genome of Synechocystis 6803 at KEGG database (https://www.genome.jp/kegg/kegg_ja.html), was synthesised by Eurofins Genomics Japan (Tokyo, Japan). The synthesised fragment was inserted within the BamHI–XhoI site of the vector pGEX6P-1 (GE Healthcare Japan, Tokyo, Japan).

The cloned expression vector was transformed in competent E. coli DH5α cells (Takara Bio, Shiga, Japan), and the transformed E. coli cells were cultivated in 5 L of Luria–Bertani medium at 30 °C with shaking at 150 rpm. Recombinant protein expression was induced overnight by adding 0.01 mM isopropyl β-D-1-thiogalactopyranoside (Wako Chemicals, Osaka, Japan) to the medium.

Affinity purification of the recombinant protein

The recombinant E. coli DH5α cells from 800 mL culture were suspended in 40 mL of phosphate-buffered saline/tween (PBST) (1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4·12H2O, 14.7 mM KH2PO4, and 0.05% Tween 20) and lysed through sonication (model VC-750; EYELA, Tokyo, Japan). The procedure was repeated 10 times for 10 s at 20% intensity. The lysed cells were centrifuged at 13,000 × g for 15 min at 4 °C. The supernatant was transferred to a 50-mL tube, and 560 µL of Glutathione Sepharose 4 B resin (GE Healthcare Japan, Tokyo, Japan) was added. Thereafter, the mixture was gently shaken for 30 min on ice. To remove the supernatant, the mixture was centrifuged at 5,800 × g for 2 min at 4 °C. The resin was re-suspended in 700 µL of PBST and washed five times. After washing, the recombinant protein was eluted with 700 µL of glutathione-S-transferase (GST) elution buffer (50 mM Tris–HCl (pH 9.6) and 10 mM reduced glutathione) five times, and the protein was concentrated using a Vivaspin 500 MWCO 50,000 device (Sartorius, Göttingen, Germany). The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). To verify protein purification, sodium dodecyl sulphate–polyacrylamide gel electrophoresis was carried out, and the gel was stained using Instant Blue reagent (Expedeon Protein Solutions, San Diego, CA, USA).

Enzyme assay

Before measuring the enzyme activity, purified 50 pmol SyAcnB was reactivated by adding 25 µL of a solution containing 5 mM dl-dithiothreitol (DTT), 100 µM Na2S, and 100 µM (NH4)2Fe(SO4)2·6H2O and incubating the mixture at 20 °C for 1 ~ 30 min. The activity of SyAcnB was measured by mixing 50 pmol holo-SyAcnB with 1 mL of the assay solution (100 mM Tris–HCl (pH 7.0–9.0) or MES-NaOH (pH 6.0–7.0) and 20 mM trisodium citrate dihydrate or 20 mM dl-isocitrate trisodium salt hydrate). The enzymatic reaction was initiated by adding reactivated SyAcnB. The formation of cis-aconitate was monitored by measuring the absorbance at 240 nm using a Hitachi U-3310 spectrophotometer (Hitachi High-Tech, Tokyo, Japan)51. One unit of SyAcnB activity was defined as the formation of 1 µmol cis-aconitate per minute. Unit/mg represents the value of one unit divided by the amount of purified protein (mg). The Km and Vmax values were calculated using curve fitting of Michaelis–Menten equation with the KaleidaGraph ver. 4.5 software and the kcat values were calculated from Vmax values. The 44 amino acid sequence of AcnSP from Synechocystis 6803 was synthesized by Eurofins Genomics Japan (Tokyo, Japan) with a purity of 91.6%.

Statistical analysis

Paired two-tailed Student's t-tests were performed to calculate the P-values using Microsoft Excel for Windows (Redmond, WA, USA). All experiments were independently carried out three times.