Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions. Supplementary Information The online version contains supplementary material available at 10.1007/s11103-023-01401-0.

The phylogenetic analysis of the Pyks of Synechocystis revealed bacterial Pyk and an evolutionary distance between the two isoforms of Pyks, Pyk1 (hereafter, SyPyk1) and Pyk2, in cyanobacteria (Haghighi 2021).Bacterial Pyk is classified as PykA and PykF, and sugar monophosphates, such as AMP, G6P, and R5P, stimulate PykA.PykF is activated by sugar diphosphates, such as FBP, in Escherichia coli (Kornberg and Malcovati 1973;Waygood et al. 1975Waygood et al. , 1976)).SyPyk2 is classified into pykF, and pyk2 knockout causes severe growth defects (Yao et al. 2020).In a previous study, the biochemical analysis of the Pyk of another cyanobacterium, Synechococcus sp.PCC 6301 (hereafter, Synechococcus Pyk), was reported to be homologous to that of Synechocystis pyk2, suggesting that ATP and TCA metabolism inhibited Synechococcus Pyk, thus indicating its roles under dark conditions (Knowles et al. 2001).pyk2 expression increases in the wild-type strain in the presence of glucose (Kaniya et al. 2013).pyk2 expression patterns remain unchanged during the day/night cycle (Saha et al. 2016).These reports indicate that SyPyk2 is essential during photosynthetic and nonphotoautotrophic conditions in Synechocystis.This study revealed the regulatory properties of SyPyk2 via biochemical analysis and demonstrated that sugar phosphates activated SyPyk2 activity.

Construction of cloning vector for recombinant SyPyk2
The amino acid sequence of Pyk2 (sll1275) polypeptide was obtained from the Kyoto Encyclopedia of Genes and Genomes database (https:// www.genome.jp/ kegg/) and synthesized by Eurofins Genomics Japan (Tokyo, Japan).The synthesized fragment was inserted within the BamHI-XhoI site of the vector pGEX6P-1 (GE Healthcare Japan, Tokyo, Japan).The cloned expression vector was transformed into competent E. coli BL21 cells (Takara Bio, Shiga, Japan) and cultured in 6 mL of Luria-Bertani medium at 30 °C with shaking at 150 rpm.Recombinant E. coli BL21 cells from 1.2-L culture were suspended in 40 mL of phosphate-buffered saline/Tween (PBST) (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) and sonicated (model VC-750; EYELA, Tokyo, Japan).This procedure was repeated 10 times at 20% intensity for 20 s.The lysed cells were centrifuged twice at 12,500 rpm for 15 min at 4 °C.The supernatant was transferred into a 50 mL tube, and 2 mL of Glutathione Sepharose 4 B resin (Cytiva Japan, Tokyo, Japan) was added.The tubes were shaken gently for 60 min on ice.The mixture was centrifuged at 8,000 rpm for 2 min at 4 °C to remove the supernatant.The resin was transferred to a 15 mL tube and washed using PBST.After washing, the recombinant protein was eluted five times using 650 μL of glutathione-S-transferase (GST) elution buffer (50 mM Tris-HCl, pH 9.6-, and 10-mM reduced glutathione) and incubated in Vivaspin 500 MWCO 50,000 device (Sartorius, Göttingen, Germany) for protein concentration.The protein concentrations were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA).The solution was transferred to a 1.5 mL tube, and 40 units of PreScission Protease (equivalent to the purified recombinant protein) (Cytiva) were added and allowed to stand at 4 °C for 16 h to remove GST-tag from the recombinant proteins.Approximately 750 µL of Glutathione Sepharose 4B resin was added, rotated for 1 h at room temperature to remove the cleaved tag from the solution, at 11,000 rpm for 4 min at 4 °C, and the supernatant was collected.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to confirm protein purification, and gels were stained using Quick Blue reagent (Bio-Dynamics Inc.Tokyo, Japan).

Statistical analyses
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 conducted independently in triplicate.

Purification of SyPyk2 and determination of optimal temperature and pH
We expressed GST-tagged SyPyk2 proteins in E. coli BL21 and purified them using affinity chromatography (Fig. 2a).SyPyk2 activity for PEP was the highest in MES-NaOH buffer at pH 7.0 and temperature 55 °C (Fig. 2b and c).The experiments measured at pH 8.5 and 9.0 Tri-HCl using Mn 2+ was precipitated (Fig. 2b).Following this, SyPyk2 activities for PEP were measured under optimal conditions (55 °C and pH 7.0) or intracellular conditions (30 °C and pH 7.8).

Dependence SyPyk2 cations for catalytic activity
Similar to the other bacterial Pyks (Waygood and Sanwal 1974;Kapoor and Venkitasubramanian 1983;Wu and Turpin 1992;Snášel and Pichová 2019), SyPyk2 activity was dependent on divalent cations such as Mg 2+ or Mn 2+ , and the V max (maximum reaction velocity) of SyPyk2 activity in the presence of Mn 2+ was half of that in the presence of Mg 2+ (Figs. 3, 4a, and b).The activity of SyPyk2 was higher in the presence of divalent cations than in the presence of monovalent cations, and its activation by monovalent cations was not K + -specific (Fig. 3).We determined the kinetic parameters of SyPyk2 with respect to its dependence on Mg 2+ and Mn 2+ .Under optimal conditions, the K m (half-saturation concentration) value of SyPyk2 for Mg 2+ and Mn 2+ dependence was 3.54 ± 0.61 and 0.296 ± 0.02 mM, respectively (Fig. 4a and b).Under intracellular conditions, the K m value of SyPyk2 for Mg 2+ and Mn 2+ dependence was 6.70 ± 0.26 and 2.18 ± 0.51 mM, respectively (Fig. 4a and  b).The K m value of Synechococcus Pyk and Synechocystis PEPC for Mg 2+ dependence were 2.9 and 4.27 ± 0.46 mM, respectively (Knowles et al. 2001;Scholl et al. 2020).Thus, we defined that the optimum conditions for SyPyk2 were 15 mM MgCl 2 and 5 mM MnCl 2 .

Determination of kinetic parameters of SyPyk2
We measured the kinetic parameters of SyPyk2 for PEP and ADP under optimal conditions.The saturation curves of SyPyk2 for PEP displayed a sigmoidal curve with V max and K m of 241 ± 10.5 unit/mg and 1.53 ± 0.07 mM, respectively, and a Hill coefficient of 3.10 ± 0.11, indicating positive homotropic cooperativity (Fig. 5a and Table 1).The saturation curves of SyPyk2 for ADP followed a hyperbolic (Michaelis-Menten) curve with V max and K m of 239 ± 6 unit/mg and 0.0527 ± 0.0075 mM, respectively (Fig. 5b and Table 1).The saturation curves of SyPyk2 for PEP and ADP under intracellular conditions were determined.The saturation curves of SyPyk2 for PEP showed a sigmoidal curve with V max and K m of 119 ± 7 unit/mg and 2.54 ± 0.12 mM, respectively, and a Hill coefficient of 2.60 ± 0.18, and Mn 2+ , respectively.Blue and green represented the buffer MES-NaOH and Tris-HCl, respectively.The concentrations of phospho-enolpyruvate (PEP), adenosine diphosphate (ADP), and KCl were fixed at 5.0, 2.0, and 100 mM, respectively.The experiments of Mn 2+ measured at pH 8.5 and 9.0 Tri-HCl was precipitated.The mean ± SD values were calculated from three independent experiments.c The effects of temperature on SyPyk2 activity.This experiment was measured in MES-NaOH buffer pH 7.0, and 15 mM Mg 2+ of the cofactor was used.PEP, ADP, and KCl concentrations were fixed at 5.0, 2.0, and 100 mM, respectively.The mean ± SD values were calculated from three independent experiments Fig. 3 Effects of cofactor monovalent and divalent cations for Synechocystis pyruvate kinase 2 (SyPyk2) activity.The monovalent and divalent cations were fixed at 100 and 5 mM, respectively, except for MgCl 2 and ZnCl 2 fixed at 15 and 0.5 mM.The experiment was performed using 100 mM MES-NaOH buffer (pH 7.0) at 55 °C.The concentrations of PEP and ADP were fixed at 5.0 and 2.0 mM, respectively.The mean ± SD values were calculated from three independent experiments.K, KCl; Na, NaCl 2 ; NH 4 , NH 4 Cl; Mg, MgCl 2 •6H 2 O; Mn, MnCl 2 •4H 2 O; Ca, CaCl 2 ; Zn, ZnSO 4 •7H 2 O suggesting positive homotropic cooperativity (Fig. 5a and Table 1).The saturation curves of SyPyk2 for ADP exhibited a hyperbolic curve with V max and K m of 80.3 ± 5.3 Unit/mg and 0.0602 ± 0.0081 mM, respectively (Fig. 5b and Table 1).Similar to Synechococcus Pyk and other bacterial Pyks, the saturation curves of SyPyk2 for PEP showed sigmoidal curves (Knowles et al. 2001;Jetten et al. 1994;Abdelhamid et al. 2019;2021), and for ADP, hyperbolic curves (Abdelhamid et al. 2019;2021).Additionally, we measured the activity of SyPyk2 at 30 °C and pH 7.0; the conditions were optimal for Synechocystis PEPC (Takeya et al. 2017) and competed with SyPyk2 for PEP consumption.The saturation curves of SyPyk2 for PEP displayed a sigmoidal curve with V max and K m of 132 ± 5 unit/mg and 2.36 ± 0.2 mM, respectively, and a Hill coefficient of 2.61 ± 0.18, indicating positive homotropic cooperativity and intracellular conditions (Supplemental Fig. 1a and Table 2).The saturation curves of ADP followed a hyperbolic curve with V max and K m of 123 ± 9 unit/mg and 0.111 ± 0.017 mM, respectively (Supplemental Fig. 1b and Table 2).In these conditions, the K m value of SyPyk2 for PEP was approximately 30-, 40-, or 20-fold higher than that of SyPyk2 for ADP under optimum, intracellular, and optimum for Synechocystis PEPC conditions, respectively (Tables 1 and 2).The K m value of SyPyk2 for ADP was half of that of Synechococcus Pyk, and the K m value of SyPyk2 for PEP was 5-fold higher than that of Synechococcus Pyk (Knowles et al. 2001).Compared with the K m value of Synechocystis PEPC (0.34 mM: Takeya et al. 2017, 0.88 mM: Scholl et al. 2020) for PEP, SyPyk2 required more than 2-fold PEP (Tables 1 and 2).

Activation and inhibition of SyPyk2 by sugar phosphates and organic acids
We measured the relative catalytic activity of SyPyk2 for PEP in the presence of sugar phosphates from the EMP/ gluconeogenesis and OPP pathways at 1 mM, and PEP and ADP was fixed at K m (2.5 mM) and 2 mM respectively, under intracellular conditions.The effectors did not affect LDH (coupled enzyme) used in the experiment.Additionally, several effector metabolites known to inhibit Pyk from the TCA cycle, such as citrate and 2OG, were added at 1 mM under optimum conditions (Wu and Turpin 1992;Knowles et al. 2001).Under optimal conditions, SyPyk2 was activated by 1 mM G6P and R5P up to 200 and 150%, respectively, and inhibited down to 75% by ATP (Fig. 6).Under intracellular conditions, SyPyk2 was activated in the presence of 1 mM G6P and R5P by 150 and 125%, respectively, and inhibited in the presence of ATP by up to 75% (Fig. 6).Unlike Synechococcus Pyk and green alga, Chlamydomonas reinhardtii Pyk, SyPyk2 was not activated by AMP or F6P and not inhibited by the TCA cycle metabolites, such as citrate, 2OG, and malate (Fig. 6; Wu and Turpin 1992;Knowles et al. 2001).
Furthermore, we calculated the kinetic parameters of SyPyk2 for PEP in the presence of activators or inhibitors under intracellular conditions.The saturation curves of SyPyk2 for PEP in the presence of G6P revealed a hyperbolic curve with V max and K m of 122 ± 5 unit/mg and 0.607 ± 0.01 mM, respectively, and a Hill coefficient The kinetic parameters of the activity of Synechocystis pyruvate kinase 2 (SyPyk2) in the presence of activators (glucose-6-phosphate, G6P and ribose-5-phosphate, R5P) or inhibitor (adenosine triphosphate, ATP) and coexisting metabolites.All experiments were performed under intracellular conditions of 30 °C and pH 7.8 in Tris-HCl buffer, except for those marked as 55 °C and pH 7.0 in MES-NaOH buffer.Each kinetic parameter is explained as follows: V max (maximum reaction velocity); K m , half-saturation concentration; k cat , turnover number; k cat /K m , catalytic efficiency; n H , Hill coefficient.Mean ± SD values were calculated from three independent experiments of 2.0 ± 0.1, indicating G6P converting SyPyk2 from sigmoidal to hyperbolic kinetics (Fig. 7a and Table 1).The enzymatic activities of SyPyk2 for PEP in the presence of R5P displayed a hyperbolic curve with V max and K m of 125 ± 18 unit/mg and 0.548 ± 0.132 mM, respectively, and a Hill coefficient of 1.4 ± 0.2, indicating R5P altering SyPyk2 from sigmoidal to hyperbolic kinetics (Fig. 7a and Table 1).Similar to Synechococcus Pyk, G6P and R5P decreased the K m value of SyPyk2 for PEP to one-fifth of its value (Knowles et al. 2001 and Table 1).Moreover, the K m value of SyPyk2 was increased by ATP from 2.54 to 2.73 mM (Fig. 7b and Table 1).To demonstrate the effects of ATP for SyPyk2, we calculated the kinetic parameters of SyPyk2 for PEP in the presence of ATP and either G6P or R5P under intracellular conditions (Fig. 7a and b).G6P, R5P, and ATP concentrations were fixed at 1 mM.The saturation curves of SyPyk2 for PEP in the presence of ATP and G6P revealed a hyperbolic curve with V max and K m of 130 ± 29.5 unit/mg and 0.619 ± 0.022 mM, respectively, and a Hill coefficient of 1.73 ± 0.36 (Fig. 7a and b, and Table 1).The enzymatic activities of SyPyk2 for PEP in the presence of ATP and R5P displayed a hyperbolic curve with V max and K m of 84.0 ± 7.08 unit/mg and 0.572 ± 0.111 mM, respectively, and a Hill coefficient of 1.89 ± 0.41 (Fig. 7a and b and Table 1).G6P and R5P relieved the effects of ATP (Fig. 7b).Additionally, the IC 50 (median inhibition concentration) of ATP for SyPyk2 was 4.1 mM (Supplemental Fig. 2), which was approximately 3-fold higher than that of Synechococcus Pyk (1.5 mM, Knowles et al. 2001).G6P and R5P increased the affinity of SyPyk2 for PEP and remained unaltered in the presence of AMP, F6P, or FBP (Figs. 6, 7a, and b, Table 1).

Discussion
This study demonstrated the properties of SyPyk2 via biochemical analysis, with G6P and R5P increasing the affinity of SyPyk2 for PEP in vitro.The optimum pH and temperature of Pyks were discovered in bacteria (Chai et al. 2016;Kapoor and Venkitasubramanian 1983;Abbe and Yamada 1982).The optimum pH of Pyks displayed a wide peak range, from acidic to alkaline (Chai et al. 2016;Abbe and Yamada 1982).For Synechococcus Pyk, the optimal pH ranged from 6.0 to 7.8, and it was active in the dark (Knowles et al. 2001).The intracellular pH of Synechocystis under the photoautotrophic condition was 7.8 (Nakamura et al. 2021), and light to dark transition decreases the intracellular pH of other cyanobacteria from alkaline to neutral (Falkner et al. 1976;Mangan et al. 2016).Thus, the broad pH range of SyPyk2 indicated that SyPyk2 could act on PEP consumption under any cultivation conditions.The optimum temperature of SyPyk2 was 55 °C (Fig. 2c).Synechocystis grows at ~ 30 °C (Inoue et al. 2001), and the optimum temperature of SyPyk2 is higher than that of the cultivation conditions (Fig. 2c).Although for a short time 5 min, Synechocystis is viable up to 54 °C (Inoue et al. 2001).Under heat shock conditions, ATP plays a crucial role in protein maintenance through chaperones (Soini et al. 2005).The gene expression of pyk2 increases during heat shocks (Slabas et al. 2006), and hence, SyPyk2 may contribute to ATP production by increasing its enzymatic activity.
Pyks have been studied for their properties and primary sequences (Hunsley and Suelter 1969;Cottam et al. 1969;Abdelhamid et al. 2019;2021).All Pyks require divalent cations, such as Mg 2+ or Mn 2+ , and numerous Pyks require K + for activity (Baek and Nowak 1982;Kachmar and Boyer 1953).SyPyk2 showed Mg 2+ -and Mn 2+ -dependent activity and other bacterial Pyks, and its activity was stimulated by monovalent cations, such as K + , Na + , or NH 4 + (Fig. 3).Bacterial Pyks are classified into two types: PykA, which is stimulated by sugar monophosphates, such as AMP, G6P, and R5P, and PykF, activated by sugar diphosphates, such as FBP in E. coli (Kornberg and Malcovati 1973;Waygood et al. 1975Waygood et al. , 1976)).Moreover, SyPyk2 is classified as PykF (Kaneko et al. 1996).In silico analysis suggests that two isozymes, Pyk1 and Pyk2 have the same allosteric sites for G6P, R5P, FBP, AMP, and ATP (Haghighi 2021).Pyks containing Pyk1 and Pyk2 from Synechocystis cells are activated by G6P, F6P, R5P, and AMP but not by FBP (Knowles and Plaxton 2003).Based on these findings, we demonstrated the regulation of SyPyk2 by adding various metabolites from the OPP pathway, EMP/gluconeogenesis pathway, and TCA cycle (Fig. 7a).Our findings reveal a decrease in the K m value of SyPyk2 with K-type characteristics, indicative of altered substrate affinity and allosteric activation by G6P and R5P (Fig. 7b and Table 1).Our results suggest that G6P and R5P may also activate SyPyk1.Therefore, SyPyk2 is dependent on divalent cations, such as Mg 2+ and Mn 2+ , and is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP.
The K m values of SyPyk2 for PEP were 40-fold higher than ADP, indicating a higher requirement for PEP in its enzymatic reaction than ADP under intracellular conditions (Table 1).The K m value of Synechococcus Pyk for PEP is higher than that for ADP (Knowles et al. 2001).The K m value of SyPyk2 for PEP is higher than Synechococcus Pyk 5-fold (Table 1 and Knowles et al. 2001).Additionally, the absolute concentration of ADP in Synechocystis cells is three times higher than that of PEP (Dempo et al. 2014).These data indicate that these two Pyk enzymes, Synechocystis and Synechococcus, are limited by PEP concentration in their reactions under photosynthetic conditions.However, its mechanism is different.Synechococcus Pyk has high PEP affinity and allosteric inhibition by citrate and ATP (Knowles et al. 2001).Synechocystis has a low affinity and not inhibited by either citrate or ATP (Table 1 and Fig. 6).Thus, these findings suggested that the availability of PEP limited the enzymatic activity of SyPyk2 for the flux of PEP to pyruvate via SyPyk2 under photosynthetic conditions.
Rapid glycogen catabolism induces glucan polymer such as G6P and signaling metabolites such as 2OG occur during nitrogen depletion (Joseph et al. 2014), indicating that G6P may activate SyPyk2.Although Synechococcus Pyk is repressed by 2OG, SyPyk2 is not (Fig. 6).However, a previous study reveals that pyk1 expression is induced 3.5-fold, and pyk2 expression is reduced by half during nitrogen-deficient conditions (Osanai et al. 2006).Hence, these findings indicate that to provide a carbon source to the TCA cycle for 2OG production, SyPyk2 may act during the initial stages of nitrogen depletion through G6P and R5P activation and then be replaced with SyPyk1.Furthermore, pyk1 is regulated by several nitrogen-related regulators such as SigE, Rre37 thorough with NtcA (Giner-Lamia et al. 2017;Iijima et al. 2014;Osanai et al. 2005).Therefore, in Synechocystis, we consider SyPyk1 and SyPyk2 to mainly function as pyruvate kinase during the late and initial nitrogen depletion stages, respectively.
In comparison to the K m value of PEP for Synechocystis PEPC (0.34 mM: Takeya et al. 2017;0.88 mM: Scholl et al. 2020 and Table 2), SyPyk2 (2.54 mM, Table 1) required more than 2-fold higher concentration of PEP (Tables 1 and  2).The K m value of SyPyk2 for PEP was decreased from 2.54 to 0.607 or 0.548 mM by G6P or R5P, respectively (Fig. 7a and b and Table 1).The higher PEP requirement and the enhanced affinity of SyPyk2 for PEP by G6P and R5P, suggesting a role for SyPyk2 in Synechocystis cells.In a previous study, Pyk from Synechocystis demonstrated a higher Pyk activity under heterotrophic conditions than under photoautotrophic and mixotrophic conditions (Knowles and Plaxton 2003).Recently, ME, which generates pyruvate from malate by the ME-dependent TCA cycle, was reportedly active under photoautotrophic conditions (Katayama et al. 2022), indicating that pyruvate is synthesized by ME and not by Pyk (Bricker et al. 2004;Qian et al. 2018).The pathway involving PEPC, MDH, and ME constitutes an alternate route for pyruvate formation in Synechocystis cells under photosynthetic conditions (You et al. 2014;Bricker et al. 2004).ATP functions as an inhibitor of Synechococcus Pyk, which is the homolog of SyPyk2 (Knowles et al. 2001).This observation suggests that the lack of Pyk flux under photosynthetic conditions can be attributed to ATP inhibition (Bricker et al. 2004).In E. coli, the intracellular concentration of ATP is suggested to be 0.6 mM (Boecker et al. 2019) and not much different from Synechocystis (Wan et al. 2017).G6P and ATP exhibit comparable concentrations, approximately 1.84*10 0 and 2.14*10 0 µmol/g-dry cell weight, respectively (Dempo et al. 2014).R5P is one-tenth of the concentration of ATP, amounting to 1.95*10 -1 µmol/g-dry cell (Dempo et al. 2014).Hence, to demonstrate the in vivo effects of metabolites, we maintained uniform effector concentrations at 1 mM.Inhibition by 1 mM ATP decreased the V max and increased the K m value of SyPyk2 activity (Table 1 and Fig. 7b).The V max of SyPyk2 for PEP decreased from 119 ± 7 to 107 ± 5, and the K m value of SyPyk2 for PEP increased from 2.54 to 2.74 mM (1.07-fold) (Fig. 7b and Table 1).In comparison, the K m value of Synechococcus Pyk increased from 0.54 to 0.75 mM (1.37-fold) by 0.5 mM ATP (Knowles et al. 2001).Furthermore, compared to the IC 50 of Synechococcus Pyk for ATP (1.5 mM: Knowles et al. 2001), SyPyk2 (4.1 mM: Supplemental Fig. 2) was approximately 3-fold higher, indicating that SyPyk2 is less affected by ATP than Synechococcus Pyk.Additionally, the presence of G6P and R5P alleviated the inhibitory effects of ATP, reducing the K m value of the substrate concentration to approximately onefifth (Fig. 7a and b).Our results showed that the effects of ATP on SyPyk2 are less potent than those of G6P and R5P.Considering the IC 50 and the slight increase in the K m value by ATP, it suggests that SyPyk2 is less influenced by ATP.Therefore, we conclude that the low flux of PEP to pyruvate via Pyk is due to its extremely low affinity for PEP (Tables 1  and 2; Knowles et al. 2001) and the absence of activators such as G6P and R5P under photosynthetic conditions.
The flux through the OPP pathway increases under nonphotosynthetic conditions (Wan et al. 2017) relative to photosynthetic conditions (Young et al. 2011), indicating a significant elevation in the levels of G6P and R5P under nonphotosynthetic conditions.Moreover, PEP increases and decreases under photosynthetic and nonphotosynthetic conditions, respectively (Werner et al. 2019).Consequently, the accumulation of G6P and R5P under nonphotosynthetic conditions upregulates the SyPyk2 reaction.Following SyPyk2 activation in the presence of G6P and R5P, PEP is consumed by SyPyk2, alleviating glucose-6-phosphate dehydrogenase (G6PDH encoded by zwf, slr1843) inhibition, a rate-limiting enzyme of the OPP pathway (Ito and Osanai 2020).Furthermore, PEP consumption via SyPyk2 activating relieving the inhibition of 6-phosphogluconate dehydrogenase (6PGDH encoded by gnd, sll0329) (Ito and Osanai, 2018), an enzyme involved in the third reaction of the OPP pathway (Fig. 8).In this feedforward regulation, SyPyk2 primarily acts as relieving the sugar catabolic repression under nonphotosynthetic conditions.
This study offers valuable insights into the biosynthesis and fermentation of metabolites associated with pyruvate metabolism, particularly PEP consumption in Synechocystis.
This study demonstrated that the regulation of SyPyk2 is dependent on PEP accumulation, the presence of G6P, R5P, and divalent cations, such as Mg 2+ and Mn 2+ , rather than pH and ATP.Therefore, our experiments indicated that SyPyk2 contributed less to PEP consumption under photosynthetic conditions and that it plays a pivotal role in sugar catabolism under nonphotosynthetic conditions in response to sugar phosphate accumulation.

Fig. 2
Fig. 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and optimal pH and temperature for Synechocystis pyruvate kinase 2 (SyPyk2).a Purified GST-tagged SyPyk2 (89 kDa) and untagged SyPyk2 (63 kDa) proteins.GST-Pyk2 indicated GST-tagged SyPyk2, and Pyk2 indicated untagged SyPyk2.The gel was prepared using 8% (w/v) acrylamide and stained with Quick Blue G250.Optimum pH and temperature for SyPyk2.b Effects of the pH on SyPyk2 activity.The circle and square represented Mg 2+ and Mn 2+ , respectively.Blue and green represented the buffer MES-NaOH and Tris-HCl, respectively.The concentrations of phospho-

Fig. 4
Fig. 4 Synechocystis pyruvate kinase 2 (SyPyk2) activity at different concentrations of MgCl 2 (a) and MnCl 2 (b).These experiments were performed under optimum conditions at 55 °C and pH 7.0 in MES-NaOH buffer (blue) or intracellular conditions at 30 °C and pH 7.8

Table 1
Kinetic parameters of pyruvate kinase 2 under optimal and intracellular conditions