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

Abstract

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 K_m 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.

Keywords: Synechocystis sp. PCC 6803; Glycolysis; Microalgae; Pyruvate kinase.

Fig. 1

Pathway map of Synechocystis sp. PCC 6803 (Synechocystis). The metabolic maps of the Embden–Meyerhof–Parnas (EMP) pathway/gluconeogenesis, oxidative pentose phosphate (OPP) pathway, pyruvate metabolism, and tricarboxylic acid (TCA) cycle. The gene names encoding metabolic enzymes in Synechocystis were obtained from the Kyoto Encyclopedia of Genes and Genomes database. The rounded rectangle indicated value-added metabolites from Synechocystis

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 Mg²⁺ and Mn²⁺, respectively. Blue and green represented the buffer MES-NaOH and Tris–HCl, respectively. The concentrations of phosphoenolpyruvate (PEP), adenosine diphosphate (ADP), and KCl were fixed at 5.0, 2.0, and 100 mM, respectively. The experiments of Mn²⁺ 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 Mg²⁺ 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 MgCl₂ and ZnCl₂ 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, NaCl₂; NH₄, NH₄Cl; Mg, MgCl₂·6H₂O; Mn, MnCl₂·4H₂O; Ca, CaCl₂; Zn, ZnSO₄·7H₂O

Fig. 4

Synechocystis pyruvate kinase 2 (SyPyk2) activity at different concentrations of MgCl₂ (a) and MnCl₂ (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 in Tris–HCl buffer (yellow). These experiments fixed the phosphoenolpyruvate (PEP), adenosine diphosphate (ADP), and KCl concentrations at 5.0, 2.0, and 100 mM, respectively. The mean ± SD values were calculated from three independent experiments

Fig. 5

Saturation curves of Synechocystis pyruvate kinase 2 (SyPyk2) for phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP). a The saturation curves of SyPyk2 for PEP. These measurements were performed in an optimum condition at 55 °C and pH 7.0 in MES-NaOH buffer (blue) or an intracellular condition of 30 °C and pH 7.8 in Tris–HCl (yellow). The ADP concentration was 2.0 mM. The mean ± SD values were calculated from three independent experiments. b The saturation curves of SyPyk2 for ADP. These measurements were performed in an optimum condition at 55 °C and pH 7.0 in MES-NaOH buffer (blue) or an intracellular condition of 30 °C and pH 7.8 in Tris–HCl (yellow). The PEP concentration was 5 mM. The concentrations of KCl and MgCl₂ were 100 and 15 mM, respectively. The mean ± SD values were calculated from three independent experiments

Fig. 6

Effects of effectors for Synechocystis pyruvate kinase 2 (SyPyk2) activity. The effects of various metabolites on the activity SyPyk2. These experiments measured optimum conditions at 55 °C and pH 7.0 in MES-NaOH buffer (left blue bar) and intracellular conditions at 30 °C and pH 7.8 in Tris–HCl buffer (right yellow bar). The mean ± SD values were calculated from three independent experiments. The concentration of PEP and ADP were fixed at K_m (2.5 mM) and 2.0 mM, respectively. In the measurements of the saturation curves of SyPyk2, the concentrations of KCl and MgCl₂ were 100 and 15 mM, respectively. The concentration of several effectors was 1.0 mM. G6P, glucose-6-phosphate-2Na; F6P, fructose-6-phosphate-2Na; FBP, fructose-1, 6-bisphosphate-3Na; R5P, ribose-5-phosphate-2Na; 6PG, 6-phospho-D-gluconate; AMP, adenosine monophosphate-Na; ADP, adenosine diphosphate-2Na; ATP, adenosine triphosphate-2Na; Cit, citrate-3Na; 2OG, 2-oxoglutarete; Suc, succinate-2Na; Fum: fumarate, Mal: malate-Na. The asterisks indicated significant differences between the absence and presence of the salt (Student’s t-test; *P < 0.01, **P < 0.005)

Fig. 7

a Influence of several effectors for Synechocystis pyruvate kinase 2 (SyPyk2) activity. Circles (blue) indicated the phosphoenolpyruvate (PEP) saturation curve, squares (red and purple) indicated 1.0 mM of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P), diamonds (gray) indicated 1.0 mM of adenosine triphosphate (ATP) added, and red or purple diamonds indicated the presence of G6P and R5P with ATP added respectively. The mean ± SD values were calculated from three independent experiments. b This figure shows the K_m value of SyPyk2 for PEP in the presence of G6P, R5P, and ATP, coexisting intracellular conditions of 30 °C and pH 7.8 in Tris–HCl buffer. The mean ± SD values were calculated from three independent experiments

Fig. 8

Model of glucose-6-phosphate (G6P), ribose-5-phosphate (R5P) and phosphoenolpyruvate (PEP) regulation of carbon flow under photosynthetic or nonphotosynthetic conditions in Synechocystissp. PCC 6803. EMP, Embden–Meyerhof–Parnas pathway; OPP, oxidative pentose phosphate pathway; CBB, Calvin Benson Bassham cycle; Pyr, pyruvate; G6PDH, glucose-6-phosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; Pyk2, pyruvate kinase 2