Metabolite production from carbon dioxide using sugar catabolism in cyanobacteria has been in the spotlight recently. Synechocystis sp. PCC 6803 (Synechocystis 6803) is the most studied cyanobacterium for metabolite production. Previous in vivo analyses revealed that the oxidative pentose phosphate (OPP) pathway is at the core of sugar catabolism in Synechocystis 6803. However, the biochemical regulation of the OPP pathway enzymes in Synechocystis 6803 remains unknown. Therefore, we characterized a key enzyme of the OPP pathway, glucose-6-phosphate dehydrogenase (G6PDH), and related enzymes from Synechocystis 6803. Synechocystis 6803 G6PDH was inhibited by citrate in the oxidative tricarboxylic acid (TCA) cycle. Citrate has not been reported as an inhibitor of G6PDH before. Similarly, 6-phosphogluconate dehydrogenase, the other enzyme from Synechocystis 6803 that catalyzes the NADPH-generating reaction in the OPP pathway, was inhibited by citrate. To understand the physiological significance of this inhibition, we characterized succinic semialdehyde dehydrogenase (SSADH) from Synechocystis 6803 (SySSADH), which catalyzes one of the NAD(P)H generating reactions in the oxidative TCA cycle. Similar to isocitrate dehydrogenase from Synechocystis 6803, SySSADH specifically catalyzed the NADPH-generating reaction and was not inhibited by citrate. The activity of SySSADH was lower than that of other bacterial SSADHs. Previous and this studies revealed that unlike the OPP pathway, the oxidative TCA cycle is a pathway with low efficiency in NADPH generation in Synechocystis 6803. It has, thus, been suggested that to avoid NADPH overproduction, the OPP pathway dehydrogenase activity is repressed when the flow of the oxidative TCA cycle increases in Synechocystis 6803.

Introduction

Cyanobacteria are photoautotrophic bacteria that perform photosynthesis to generate oxygen and produce various metabolites from carbon dioxide. Thus far, many researchers have studied carbon anabolism in cyanobacteria. In recent years, global warming and depletion of fossil fuels have been a cause of great concern. Therefore, the production of bioplastics and biofuels from carbon dioxide using sugar catabolism in cyanobacteria has been widely studied [1,2]. However, the regulation mechanism of the sugar catabolism in cyanobacteria is not well known, so that studies on sugar catabolism in cyanobacteria have been required for metabolic engineering of cyanobacteria. Among cyanobacteria, the model cyanobacterium, Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803), which has several advantages as a model organism, such as complete genomic information [3], rapid growth, and natural transformation capability [4], has been especially studied as a host for the production of various useful substances [2,5].

Sugar catabolic pathway consists of upstream pathways such as the glycolysis, Entner–Doudoroff pathway and oxidative pentose phosphate (OPP) pathway, and a downstream pathway, the tricarboxylic acid (TCA) cycle. The composition of the cyanobacterial sugar catabolic pathway is unique. The TCA cycle is preserved in almost all cyanobacteria including Synechocystis 6803, but unlike other organisms, cyanobacteria use 2-oxoglutarate (2OG) decarboxylase and succinic semialdehyde dehydrogenase (SSADH, EC 1.2.1.79) to convert 2OG to succinate [6]. Also, among three upstream pathways in sugar catabolism, the OPP pathway whose enzymes generate pentoses, such as ribulose-5-phosphate, and the reducing power, NADPH (Figure 1) is an only pathway preserved in all cyanobacteria [7].

Metabolic map around the OPP pathway.

Figure 1.
Metabolic map around the OPP pathway.

The reactions in the glycolysis and OPP pathway were represented by the green and red arrowheads, respectively. The reactions consisting of several steps were represented by the dashed lines.

Figure 1.
Metabolic map around the OPP pathway.

The reactions in the glycolysis and OPP pathway were represented by the green and red arrowheads, respectively. The reactions consisting of several steps were represented by the dashed lines.

For carbon metabolic pathways, Synechocystis 6803 has characteristically more metabolites in the OPP pathway than the model heterotrophic bacterium, Escherichia coli [8]. Metabolic flux of the OPP pathway in Synechocystis 6803 markedly changes depending on the culture conditions and increases in low rather than bright light conditions [8,9]. In Synechocystis 6803, the OPP pathway is important for the generation of NADPH when photosynthesis generating NADPH is inactivated [10] and cells lacking 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44), which catalyzes the third reaction generating NADPH in the OPP pathway (Figure 1), cannot grow in dark [8]. Similarly, the Synechocystis 6803 cells lacking glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), which catalyzes the first reaction generating NADPH in the OPP pathway (Figure 1), cannot grow in low light [9]. These genetic analyses suggest that NADPH generation determines the cell growth and metabolic flux under non-photosynthetic conditions in Synechocystis 6803. Moreover, in Synechocystis 6803, the activation of gene expression of metabolic enzymes involved in the OPP pathway, promotes the production of useful substances, such as succinate, lactate, polyhydroxybutyrate, ethanol, and limonene [5,11–13]. These previous reports demonstrate that the OPP pathway is the central metabolic pathway in sugar catabolism in Synechocystis 6803. In heterotrophic bacterium and yeast, biochemical analyses demonstrated that NADPH inhibits the G6PDH and 6PGDH activities, suggesting their OPP pathway is regulated in a negative feedback manner [14,15]. In cyanobacterium Nostoc punctiforme ATCC 29133, biochemical analyses demonstrated that OpcA, a protein conserved only in cyanobacteria acts as an allosteric activator of G6PDH [16]. However, biochemical regulation determining the metabolic flux of the OPP pathway in Synechocystis 6803 has remained unknown.

In this study, we biochemically analyzed a rate-limiting enzyme in the OPP pathway, G6PDH from Synechocystis 6803 (SyG6PDH) and related metabolic enzymes and revealed the mechanism and physiological significance of biochemical regulation of the OPP pathway enzymes in Synechocystis 6803.

Experimental

Preparation of vectors

The regions containing zwf (slr1843, encoding G6PDH) and gabD (slr0370, encoding SSADH) in the Synechocystis 6803 genome were synthesized by Eurofin Genomics Japan (Tokyo, Japan) and cloned into the BamHI-XhoI site of the pGEX6P-1 vector (GE Healthcare, Little Chalfont, U.K.). Method of preparation of vector containing gnd (sll0329, encoding 6PGDH) has been described previously [17].

Purification of metabolic enzymes from Synechocystis 6803

The vectors carrying GST-tagged SyG6PDH and SySSADH were used to transform E. coli DH5α competent cells (TakaraBio). Thereafter, the transformed DH5α cells were grown in a 2 l culture at 30°C in LB media, with shaking (125 rpm). Subsequently, expression of the GST-tagged proteins were induced with 0.01 mM isopropyl β-d-1-thiogalactopyranoside (Wako Chemicals, Osaka, Japan), and the cells were grown overnight. As previously described [18], affinity purification of GST-tagged proteins was performed. Briefly, DH5α cells were disrupted by sonication (model VC-750, EYELA, Tokyo, Japan) for 200 s by 20% intensity, to release GST-tagged proteins from cells. Thereafter, the sample was centrifuged at 5800×g for 2 min at 4°C, the supernatants containing the GST-tagged proteins was transferred into two 50-ml tubes, and 560 μl of Glutathione-Sepharose 4B resin (GE Healthcare Japan, Tokyo, Japan) was added to each tube. Subsequently, the tubes were shaken for 30 min on ice and 1 mM ATP/MgSO4·7H2O was added to the tubes, which were then shaken for 30 min at 37°C. After the supernatant was removed by centrifugation (5800×g for 2 min at 4°C), the residual resin was washed 10 times with 700 μl of PBS-T (1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4·12H2O, 14.7 mM KH2PO4, and 0.05% Tween-20) containing 1 mM ATP/MgSO4·7H2O. The GST-tagged proteins were eluted from the resin four times using 700 μl of GST elution buffer (50 mM Tris–HCl, pH 8.0, and 10 mM reduced glutathione). After concentrating the solution containing the GST-tagged proteins, using a VivaSpin 500 MWCO 50 000 device (Sartorius, Göttingen, Germany), the solution was transferred into two 1.5 ml-tubes. To remove GST-tags from the GST-tagged proteins, seven (for SyG6PDH) or nine (for SySSADH) units of PreScission Protease (GE Healthcare Japan, Tokyo, Japan) were added to each tube, the sample was incubated for 18 h at 4°C, 600 μl of Glutathione-Sepharose 4B resin (GE Healthcare Japan, Tokyo, Japan) was added to each tube, and the mixture was rotated for 1 h at room temperature. Thereafter, the resin containing GST-tag was removed from the solution by centrifugation (5800×g for 2 min at 4°C). After purification, the concentration of protein was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Confirmation of purity of proteins was performed by SDS–PAGE using 12% SDS–PAGE gels. The staining of gels was performed using InstantBlue (Expedion Protein Solutions, San Diego, CA). Method of purification of GST-tagged Sy6PGDH had been described previously [17].

Enzyme assays

The reaction catalyzed by SyG6PDH was performed in 1 ml assay solution [50 mM potassium phosphate (pH 6.0–7.0), Tris–HCl (pH 7.0–9.0) or glycine–NaOH (pH 9.0–11.0), various concentrations of NAD(P)+, various concentrations of glucose-6-phosphate, and 8 pmol of SyG6PDH]. The reaction catalyzed by Sy6PGDH was performed in 1 ml assay solution [50 mM Tris–HCl (pH 7.3), various concentrations of NADP+, various concentrations of 6-phosphogluconate, and 16 pmol of Sy6PGDH]. The reaction catalyzed by SySSADH was performed in 1 ml assay solution [50 mM glycine-NaOH (pH 8.6–11.0), various concentrations of NADP+, various concentrations of succinic semialdehyde, 2 mM MgCl2, and 40 pmol of SySSADH]. Enzymatic activities were calculated from the change in absorbance at A340 and monitored by a Hitachi U-3310 spectrophotometer (Hitachi High-Tech., Tokyo, Japan). One unit of enzymatic activity represents the production volume of 1 μmol NAD(P)H per min.

Calculation of kinetic parameters

The calculation of the kinetic parameters of SyG6PDH and SySSADH was performed by curve fitting using Kaleida Graph ver. 4.5. The Hill equation (equation 1) [19] was used when evident cooperativity was confirmed. The Michaelis–Menten equation (equation 2) was used at other instances. 
v=Vmax[S]nH/([S]nH+S0.5nH)
1
 
v=Vmax[S]/([S]+Km)
2
The kcat values were calculated using the Vmax values. Vmax represents maximum reaction velocity. S0.5 and Km represent the substrate concentration at 50% Vmax, calculated using Hill equation and Michaelis–Menten equation, respectively. kcat and nH represent the turnover number and Hill coefficient, respectively. The kinetic parameters of Sy6PGDH were calculated using a Lineweaver–Burk plot, as described previously [17].

Results

Kinetic analyses of SyG6PDH

To examine the biochemical properties of SyG6PDH, we purified SyG6PDH by affinity chromatography (Figure 2a). The position of the single band obtained by SDS–PAGE was consistent with the expected molecular mass of SyG6PDH (58 kD) (Figure 2a). SyG6PDH showed consistent enzymatic activity at 20–50°C and pH 7.0–10.5 (Figure 2b,c). Under these conditions, SyG6PDH showed the highest activity at 30°C and pH 10.0 (Figure 2b,c). Therefore, we performed biochemical analyses of SyG6PDH at 30°C and pH 10.0. The S0.5 (substrate concentration at 50% Vmax), kcat (turnover number), kcat/S0.5 (catalytic efficiency), and nH (Hill coefficient) values of SyG6PDH for glucose-6-phosphate (G6P) were 25.0 ± 1.2 mM, 67.5 ± 5.0 s−1, 2.71 ± 0.32 s−1 mM−1, and 1.86 ± 0.03, respectively (Table 1). SyG6PDH used both NADP+ and NAD+ as coenzymes (Table 1). The S0.5 and kcat values of SyG6PDH for NADP+ were 0.017 ± 0.001 mM and 61.8 ± 5.7 s−1, respectively (Table 1). The S0.5 and kcat values of SyG6PDH for NAD+ were 3.94 ± 0.39 mM and 37.3 ± 3.1 s−1, respectively (Table 1). Considering the ratio of kcat/S0.5 of SyG6PDH for coenzymes, SyG6PDH was specific to NADP+ by ∼3.9 × 102-fold, compared with NAD+ (Table 1).

Temperature and pH dependence of SyG6PDH.

Figure 2.
Temperature and pH dependence of SyG6PDH.

(a) Result of SDS–PAGE after affinity purification and removal of GST-tag of SyG6PDH. (b) SyG6PDH activity under different temperature conditions. This measurement was carried out at pH 7.5. The fixed concentrations of G6P and NADP+ were 50 mM and 0.5 mM, respectively. SyG6PDH activity was represented by relative activity and the activity at 30°C was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S1. The mean ± SD was calculated using three independent data. (c) SyG6PDH activity under different pH conditions. This measurement was carried out at 30°C. The fixed concentrations of G6P and NADP+ were 50 mM and 0.5 mM, respectively. SyG6PDH activity was represented by relative activity and the activity at pH 10.0 was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S2. The mean ± SD was calculated using three independent data.

Figure 2.
Temperature and pH dependence of SyG6PDH.

(a) Result of SDS–PAGE after affinity purification and removal of GST-tag of SyG6PDH. (b) SyG6PDH activity under different temperature conditions. This measurement was carried out at pH 7.5. The fixed concentrations of G6P and NADP+ were 50 mM and 0.5 mM, respectively. SyG6PDH activity was represented by relative activity and the activity at 30°C was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S1. The mean ± SD was calculated using three independent data. (c) SyG6PDH activity under different pH conditions. This measurement was carried out at 30°C. The fixed concentrations of G6P and NADP+ were 50 mM and 0.5 mM, respectively. SyG6PDH activity was represented by relative activity and the activity at pH 10.0 was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S2. The mean ± SD was calculated using three independent data.

Table 1.
Kinetic parameters of SyG6PDH, Sy6PGDH, and SySSADH
EffectorS0.5 (Km) (mM)kcat (s−1)kcat/S0.5 (Km) (s−1 mM−1)nH
SyG6PDH 
G6P None 25.0 ± 1.2 67.5 ± 5.0 2.71 ± 0.32 1.86 ± 0.03 
10 mM PEP 14.2 ± 0.8** 54.3 ± 2.8* 3.83 ± 0.39** 2.06 ± 0.26 
10 mM OAA 4.3 ± 1.5** 16.8 ± 0.9** 4.26 ± 1.50 2.58 ± 0.71 
10 mM Cit 9.4 ± 1.2** 25.0 ± 0.7** 2.70 ± 0.37  
10 mM 2OG 9.4 ± 1.6** 25.4 ± 1.1** 2.76 ± 0.61  
NADP+ None 0.017 ± 0.001 61.8 ± 5.7 3698 ± 158 1.60 ± 0.05 
10 mM PEP 0.017 ± 0.003 56.0 ± 1.1 3337 ± 513 1.27 ± 0.11 
10 mM OAA 0.0071 ± 0.0003** 19.3 ± 1.8** 2714 ± 168** 2.10 ± 0.45 
10 mM Cit 0.0088 ± 0.0007** 24.5 ± 3.1** 2798 ± 139** 1.64 ± 0.04 
10 mM 2OG 0.0080 ± 0.0005* 25.3 ± 1.8** 3169 ± 344* 2.06 ± 0.14* 
0.2 mM NADH 0.014 ± 0.001 34.7 ± 1.9* 2481 ± 276** 1.60 ± 0.40 
0.2 mM NADPH 0.076 ± 0.001** 61.3 ± 5.4 808 ± 79**  
NAD+ None 3.94 ± 0.39 37.3 ± 3.1 9.51 ± 0.86 1.34 ± 0.17 
Sy6PGDH 
6PG None [17] (Ito and Osanai., 2018) 0.052 ± 0.005 64.5 ± 8.8 1239 ± 41  
3 mM PEP 0.254 ± 0.055* 27.1 ± 0.6* 110 ± 21**  
3 mM OAA 0.048 ± 0.007* 34.5 ± 5.1* 719 ± 20**  
3 mM Cit 0.153 ± 0.018* 17.0 ± 1.7* 111 ± 5**  
3 mM 2OG 0.025 ± 0.002* 30.1 ± 0.1* 1208 ± 119  
NADP+ None [17] (Ito and Osanai., 2018) 0.058 ± 0.003 69.9 ± 5.5 1207 ± 87  
3 mM PEP 0.022 ± 0.004* 30.3 ± 1.6* 1400 ± 161  
3 mM OAA 0.026 ± 0.002* 32.3 ± 1.3* 1260 ± 105  
3 mM Cit 0.030 ± 0.008* 26.8 ± 5.2** 891 ± 78  
3 mM 2OG 0.027 ± 0.002* 30.6 ± 2.3* 1150 ± 14  
NAD+ None [17] (Ito and Osanai., 2018) 4.46 ± 0.55 4.89 ± 0.54 1.10 ± 0.08  
SySSADH 
SSA None 0.010 ± 0.001 1.38 ± 0.02 135 ± 14  
NADP+ None 0.058 ± 0.004 1.45 ± 0.10 24.9 ± 0.4  
NAD+ None ND ND ND  
EffectorS0.5 (Km) (mM)kcat (s−1)kcat/S0.5 (Km) (s−1 mM−1)nH
SyG6PDH 
G6P None 25.0 ± 1.2 67.5 ± 5.0 2.71 ± 0.32 1.86 ± 0.03 
10 mM PEP 14.2 ± 0.8** 54.3 ± 2.8* 3.83 ± 0.39** 2.06 ± 0.26 
10 mM OAA 4.3 ± 1.5** 16.8 ± 0.9** 4.26 ± 1.50 2.58 ± 0.71 
10 mM Cit 9.4 ± 1.2** 25.0 ± 0.7** 2.70 ± 0.37  
10 mM 2OG 9.4 ± 1.6** 25.4 ± 1.1** 2.76 ± 0.61  
NADP+ None 0.017 ± 0.001 61.8 ± 5.7 3698 ± 158 1.60 ± 0.05 
10 mM PEP 0.017 ± 0.003 56.0 ± 1.1 3337 ± 513 1.27 ± 0.11 
10 mM OAA 0.0071 ± 0.0003** 19.3 ± 1.8** 2714 ± 168** 2.10 ± 0.45 
10 mM Cit 0.0088 ± 0.0007** 24.5 ± 3.1** 2798 ± 139** 1.64 ± 0.04 
10 mM 2OG 0.0080 ± 0.0005* 25.3 ± 1.8** 3169 ± 344* 2.06 ± 0.14* 
0.2 mM NADH 0.014 ± 0.001 34.7 ± 1.9* 2481 ± 276** 1.60 ± 0.40 
0.2 mM NADPH 0.076 ± 0.001** 61.3 ± 5.4 808 ± 79**  
NAD+ None 3.94 ± 0.39 37.3 ± 3.1 9.51 ± 0.86 1.34 ± 0.17 
Sy6PGDH 
6PG None [17] (Ito and Osanai., 2018) 0.052 ± 0.005 64.5 ± 8.8 1239 ± 41  
3 mM PEP 0.254 ± 0.055* 27.1 ± 0.6* 110 ± 21**  
3 mM OAA 0.048 ± 0.007* 34.5 ± 5.1* 719 ± 20**  
3 mM Cit 0.153 ± 0.018* 17.0 ± 1.7* 111 ± 5**  
3 mM 2OG 0.025 ± 0.002* 30.1 ± 0.1* 1208 ± 119  
NADP+ None [17] (Ito and Osanai., 2018) 0.058 ± 0.003 69.9 ± 5.5 1207 ± 87  
3 mM PEP 0.022 ± 0.004* 30.3 ± 1.6* 1400 ± 161  
3 mM OAA 0.026 ± 0.002* 32.3 ± 1.3* 1260 ± 105  
3 mM Cit 0.030 ± 0.008* 26.8 ± 5.2** 891 ± 78  
3 mM 2OG 0.027 ± 0.002* 30.6 ± 2.3* 1150 ± 14  
NAD+ None [17] (Ito and Osanai., 2018) 4.46 ± 0.55 4.89 ± 0.54 1.10 ± 0.08  
SySSADH 
SSA None 0.010 ± 0.001 1.38 ± 0.02 135 ± 14  
NADP+ None 0.058 ± 0.004 1.45 ± 0.10 24.9 ± 0.4  
NAD+ None ND ND ND  

These measurements were carried out at optimum conditions (SyG6PDH: 30°C and pH 10.0, Sy6PGDH: 32°C and pH 7.3, and SySSADH: 45°C and pH 9.7). For the kinetic analysis of SyG6PDH, the fixed concentration of G6P or NADP+ was 100 mM or 0.5 mM, respectively. For the kinetic analysis of Sy6PGDH, the fixed concentration of 6PG or NADP+ was 2 mM. For the kinetic analysis of SySSADH, the fixed concentration of SSA or NADP+ was 1 mM. SySSADH activity using NAD+ as a coenzyme was not detected (represented by ND). The mean ± SD was calculated using three independent data. The significant differences between the absence and presence of the effector are shown by asterisks (Student's t-test; *P < 0.05, **P < 0.005).

SyG6PDH activity was affected by organic acids in the tricarboxylic acid (TCA) cycle

To find the effectors of SyG6PDH, we measured SyG6PDH activity in the presence of divalent cations and metabolites under conditions where the concentrations of G6P and NADP+ were fixed at the S0.5 values of SyG6PDH (Figure 3). SyG6PDH activity changed in the presence of various metabolites (Figure 3). In the presence of 10 mM phosphoenolpyruvate (PEP), SyG6PDH activity decreased to 78% (Figure 3). More detailed catalytic characterization showed that PEP decreased the S0.5 and kcat values of SyG6PDH for G6P, and increased the kcat/S0.5 value of SyG6PDH for G6P (Table 1).

Change of SyG6PDH activity in the presence of divalent cations and metabolites.

Figure 3.
Change of SyG6PDH activity in the presence of divalent cations and metabolites.

This measurement was carried out at 30°C and pH 10.0. The fixed concentrations of NADH and NADPH were 0.2 mM and concentrations of the other effectors were 10 mM. The fixed concentrations of G6P and NADP+ were the S0.5 values of SyG6PDH, 25.0 mM and 0.017 mM, respectively. SyG6PDH activity was represented by relative activity and the activity in the absence of effectors was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S3. The mean ± SD was calculated using three independent data. The significant differences between the absence and presence of the effector were shown by asterisks (Student's t-test; *P < 0.05, **P < 0.005). Mg: MgCl2, Ca: CaCl2, Glc: glucose, 6PG: 6-phosphogluconate, R5P: ribose-5-phosphate, FBP: fructose-1,6-bisphosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate, AcC: acetyl-CoA, OAA: oxaloacetate, Cit: citrate, 2OG: 2-oxoglutarate, SSA: succinic semialdehyde, Suc: succinate, Fum: fumarate, Mal: l-malate, Gln: l-glutamine, Glu: l-glutamate, Arg: l-arginine, and Asp: l-aspartate.

Figure 3.
Change of SyG6PDH activity in the presence of divalent cations and metabolites.

This measurement was carried out at 30°C and pH 10.0. The fixed concentrations of NADH and NADPH were 0.2 mM and concentrations of the other effectors were 10 mM. The fixed concentrations of G6P and NADP+ were the S0.5 values of SyG6PDH, 25.0 mM and 0.017 mM, respectively. SyG6PDH activity was represented by relative activity and the activity in the absence of effectors was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S3. The mean ± SD was calculated using three independent data. The significant differences between the absence and presence of the effector were shown by asterisks (Student's t-test; *P < 0.05, **P < 0.005). Mg: MgCl2, Ca: CaCl2, Glc: glucose, 6PG: 6-phosphogluconate, R5P: ribose-5-phosphate, FBP: fructose-1,6-bisphosphate, PEP: phosphoenolpyruvate, Pyr: pyruvate, AcC: acetyl-CoA, OAA: oxaloacetate, Cit: citrate, 2OG: 2-oxoglutarate, SSA: succinic semialdehyde, Suc: succinate, Fum: fumarate, Mal: l-malate, Gln: l-glutamine, Glu: l-glutamate, Arg: l-arginine, and Asp: l-aspartate.

Surprisingly, SyG6PDH activity markedly changed in the presence of metabolites in the TCA cycle which have not been reported as effectors of G6PDH (Figure 3). In the presence of 10 mM oxaloacetate (OAA), citrate (Cit), and 2OG, SyG6PDH activity decreased to 69%, 66%, and 75%, respectively (Figure 3). More detailed catalytic characterization showed that the three organic acids in the TCA cycle decreased the S0.5 and kcat values of SyG6PDH for G6P and the S0.5, kcat, and kcat/S0.5 values of SyG6PDH for NADP+ (Table 1). Similarly, at pH 6.0 and pH 8.0, SyG6PDH activity decreased in the presence of each of the three organic acids (Figure 4).

Change of SyG6PDH activity in the presence of 5 mM organic acids.

Figure 4.
Change of SyG6PDH activity in the presence of 5 mM organic acids.

This measurement was carried out at 30°C. The fixed concentrations of G6P and NADP+ were 100 mM or 0.5 mM, respectively. SyG6PDH activity was represented by relative activity and the activity in the absence of organic acids was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S4. SyG6PDH activity in the presence of 5 mM OAA, at pH 6.0, was very low and not detected (represented by ND). The mean ± SD was calculated using three independent data. The significant differences between the absence and presence of the organic acid are shown by asterisks (Student's t-test; *P < 0.05, **P < 0.005).

Figure 4.
Change of SyG6PDH activity in the presence of 5 mM organic acids.

This measurement was carried out at 30°C. The fixed concentrations of G6P and NADP+ were 100 mM or 0.5 mM, respectively. SyG6PDH activity was represented by relative activity and the activity in the absence of organic acids was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S4. SyG6PDH activity in the presence of 5 mM OAA, at pH 6.0, was very low and not detected (represented by ND). The mean ± SD was calculated using three independent data. The significant differences between the absence and presence of the organic acid are shown by asterisks (Student's t-test; *P < 0.05, **P < 0.005).

SyG6PDH activity also markedly changed in the presence of reduced forms of coenzymes, NADH and NADPH (Figure 3). In the presence of 0.2 mM NADH and NADPH, SyG6PDH activity decreased to 61% and 13%, respectively (Figure 3). More detailed catalytic characterization showed that NADH decreased the kcat and kcat/S0.5 values of SyG6PDH for NADP+ (Table 1). Also, NADPH increased the S0.5 (Km) value of SyG6PDH for NADP+, and decreased the kcat/S0.5 (Km) value of SyG6PDH for NADP+ (Table 1).

Synechocystis 6803 6PGDH activity was also affected by organic acids in the TCA cycle

To further examine the effects of the three organic acids (OAA, Cit, and 2OG) on the OPP pathway in Synechocystis 6803, we measured the sensitivity for the three organic acids of the other enzyme catalyzing the NADPH-generating reaction in the OPP pathway — 6PGDH from Synechocystis 6803 (Sy6PGDH). We used GST-tagged Sy6PGDH, as described previously [17]. Biochemical analyses of Sy6PGDH was performed at conditions optimum for Sy6PGDH (32°C and pH 7.3) [17]. In the presence of 5 mM OAA, Cit, and 2OG, Sy6PGDH activity decreased to 19%, 4%, and 35%, respectively (Figure 5). More detailed catalytic characterization showed that the inhibitory effects of the three organic acids on Sy6PGDH were different from each other (Table 1). OAA decreased the Km, kcat, and kcat/Km values of Sy6PGDH for 6PG and the Km and kcat values of Sy6PGDH for NADP+ (Table 1). Cit increased the Km value of Sy6PGDH for 6PG, and decreased the kcat and kcat/Km values of Sy6PGDH for 6PG (Table 1). Cit also decreased the Km and kcat values of Sy6PGDH for NADP+ (Table 1). 2OG decreased the Km and kcat values of Sy6PGDH for 6PG and NADP+ (Table 1). Similar to SyG6PDH, Sy6PGDH activity also decreased in the presence of PEP [17]. More detailed catalytic characterization showed that PEP increased the Km value of Sy6PGDH for 6PG, and decreased the kcat and kcat/Km values of Sy6PGDH for 6PG (Table 1). PEP also decreased the Km and kcat values of Sy6PGDH for NADP+ (Table 1).

Change of Sy6PGDH (left) and SySSADH (right) activities in the presence of 5 mM organic acids.

Figure 5.
Change of Sy6PGDH (left) and SySSADH (right) activities in the presence of 5 mM organic acids.

These measurements were carried out at optimum conditions (Sy6PGDH: 32°C and pH 7.3, SySSADH: 45°C and pH 9.7). For the measurement of Sy6PGDH activity, the fixed concentrations of 6PG and NADP+ were the Km values of Sy6PGDH, 0.052 mM and 0.058 mM [17], respectively. For the measurement of SySSADH activity, the fixed concentrations of SSA and NADP+ were the Km values of SySSADH, 0.010 mM and 0.058 mM, respectively. The enzymatic activities were represented by relative activity and the activity in the absence of organic acids was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S5. The mean ± SD was calculated using three independent data. The significant differences between the absence and presence of the organic acids are shown by asterisks (Student's t-test; *P < 0.05, **P < 0.005).

Figure 5.
Change of Sy6PGDH (left) and SySSADH (right) activities in the presence of 5 mM organic acids.

These measurements were carried out at optimum conditions (Sy6PGDH: 32°C and pH 7.3, SySSADH: 45°C and pH 9.7). For the measurement of Sy6PGDH activity, the fixed concentrations of 6PG and NADP+ were the Km values of Sy6PGDH, 0.052 mM and 0.058 mM [17], respectively. For the measurement of SySSADH activity, the fixed concentrations of SSA and NADP+ were the Km values of SySSADH, 0.010 mM and 0.058 mM, respectively. The enzymatic activities were represented by relative activity and the activity in the absence of organic acids was set at 100%. The actual enzymatic activities (Units/mg) were described in Supplementary Table S5. The mean ± SD was calculated using three independent data. The significant differences between the absence and presence of the organic acids are shown by asterisks (Student's t-test; *P < 0.05, **P < 0.005).

Biochemical analyses of Synechocystis 6803 SSADH and comparison of properties between this enzyme and two enzymes in the OPP pathway

Similar to the OPP pathway, the oxidative TCA cycle generates the reducing power. The oxidative TCA cycle includes four reductive steps, one of which generates FADH2 and the other three steps generate NAD(P)H. Among three enzymes catalyzing the NAD(P)H generating reaction in the oxidative TCA cycle in Synechocystis 6803, SSADH is the only enzyme not characterized. To understand the relationship between enzymes in the OPP pathway and the oxidative TCA cycle in Synechocystis 6803, we purified (Supplementary Figure S1a) and biochemically analyzed SSADH from Synechocystis 6803 (SySSADH). SySSADH showed the highest activity at 45°C and pH 9.7 (Supplementary Figure S1b,c; Supplementary Tables S6 and S7). Therefore, we performed biochemical analyses of SySSADH at 45°C and pH 9.7. The Km and kcat values of SySSADH for SSA were 0.010 ± 0.001 mM and 1.38 ± 0.02 s−1, respectively (Table 1). Similar to SyG6PDH and Sy6PGDH, SySSADH specifically used NADP+ as a coenzyme (Table 1). The Km and kcat values of SySSADH for NADP+ was 0.058 ± 0.004 mM and 1.45 ± 0.010 s−1, respectively (Table 1). The kcat/S0.5 (Km) value of SySSADH for NADP+ (24.9 mM−1) was 0.7% of that of SyG6PDH (3698 s−1 mM−1) and 2.1% of that of Sy6PGDH (1207 s−1  mM−1), respectively (Table 1). SySSADH activity did not decrease in the presence of the three organic acids, which decreased SyG6PDH and Sy6PGDH activity (Figure 5).

Discussion

In this study, we carried out biochemical analyses of critical enzymes in the OPP pathway in Synechocystis 6803 and revealed the unique biochemical regulation of this pathway.

The S0.5 (Km) value of SyG6PDH for G6P (25.0 mM) was markedly higher than that of G6PDHs from heterotrophic bacteria (10 species: 0.019–3 mM) [14,20–28] (Table 1). The S0.5 (Km) value of G6PDH from cyanobacterium, Nostoc punctiforme ATCC 29133 for G6P drastically decreases from 65 to 1.9 mM in the presence of OpcA [16]. Moreover, SyG6PDH activity in mutant cells lacking OpcA is lower compared with SyG6PDH activity in wild type cells, in the presence of G6P, whose concentration is less than the saturated concentration of SyG6PDH [29]. This suggests that the affinity of SyG6PDH for G6P is appropriately regulated by OpcA in vivo. Similar to Sy6PGDH [17], SyG6PDH specifically showed enzymatic activity for NADP+ and not NAD+ (Table 1). The determination of absolute metabolite concentrations (μmol/g-drycell weight) in Synechocystis 6803 indicates that Synechocystis 6803 has as much NAD+ as NADP+ [30]. This suggests that in addition to Sy6PGDH [17], SyG6PDH specifically uses NADP+ as a coenzyme in vivo. The S0.5 (Km) value of SyG6PDH for NADP+ (0.017 mM) was within the range of those of bacterial G6PDHs (12 species: 0.005–0.130 mM) [14,16,20–28,31] and approximately the same as that of G6PDH from E. coli (0.015 mM) [28] (Table 1). Moreover, the S0.5 (Km) value of SyG6PDH for NADP+ (0.017 mM) was lower than that of Sy6PGDH (0.058 mM) [17] (Table 1). NADP+ might be preferentially utilized by SyG6PDH rather than Sy6PGDH in vivo. The kcat values of SyG6PDH (67.5 s−1 for G6P and 61.8 s−1 for NADP+) were within the range of those of bacterial G6PDHs (six species: 44.1–540.0 s−1) [24–27,31,32] and approximately the same as that of G6PDH from Thermotoga maritima (72.3 s−1) [25] (Table 1). A comparison of kinetic parameters among bacterial 6PGDHs had previously revealed that the activity of Sy6PGDH is markedly higher than that of other bacterial 6PGDHs [17]. A comparison of kinetic parameters among bacterial G6PDHs in this study revealed that the activity of SyG6PDH is not much different from that of other bacterial G6PDHs.

Similar to Sy6PGDH [17], SyG6PDH activity was regulated by metabolites rather than divalent cations (Figure 3). Among metabolites which decreased SyG6PDH activity, NADPH, PEP, OAA, Cit, and 2OG also decreased Sy6PGDH activity [17] (Figures 3 and 5). Kinetic analysis of SyG6PDH in the presence of NADPH demonstrates that NADPH is a competitive inhibitor for NADP+ of SyG6PDH and other G6PDHs [33] (Table 1). Namely, SyG6PDH activity is regulated by the NADPH/NADP+ ratio. In Synechocystis 6803, deficiency of the gene encoding NAD kinase led to extremely high NADPH/NADP+ ratio and growth retardation [34], suggesting that extremely high NADPH/NADP+ ratio has negative effect on the cell growth. Therefore, it is expected that the inhibition of SyG6PDH by NADPH is necessary to avoid NADPH overproduction. In 12-h light/dark cycle, the NADPH/NADP+ ratio increases under the early light period where the light reaction of photosynthesis becomes active [35]. The OPP pathway flux under photoautotrophic conditions [36] is low relative to that under dark heterotrophic conditions [8]. These suggest that the OPP pathway flux under photoautotrophic conditions is repressed by low SyG6PDH activity caused by high NADPH/NADP+ ratio. As for Sy6PGDH, NADPH is a non-competitive inhibitor for NADP+ [17]. In the presence of PEP, which is a potent inhibitor of bacterial G6PDH [37], the kcat value of SyG6PDH decreased (Table 1). Kinetic analyses of Sy6PGDH in the presence of PEP demonstrate that PEP is a mixed inhibitor and an uncompetitive inhibitor for 6PG and NADP+ of Sy6PGDH, respectively (Table 1). PEP accumulates in Synechocystis 6803 cells under light conditions rather than dark conditions [38]. Among cyanobacteria, Synechocystis 6803 particularly accumulate PEP in the cell [30]. This suggests that the activity of the OPP pathway enzymes in Synechocystis 6803 is repressed by PEP under light conditions, where Synechocystis 6803 produces NADPH via photosynthesis. Among the three organic acids (OAA, Cit, and 2OG) which decreased SyG6PDH and Sy6PGDH activity, OAA is unstable [39], so that the absolute concentration (μmol/g-drycell weight) of OAA in Synechocystis 6803 is not determined [30]. Therefore, the inhibitory effects of SyG6PDH and Sy6PGDH by OAA are uncertain in vivo. The inhibitory effect of Sy6PGDH by Cit was larger than that of Sy6PGDH by OAA and 2OG, and Cit decreased not only the kcat value but also the affinity for 6PG of Sy6PGDH unlike OAA and 2OG (Figure 5 and Table 1). Cit also shows the inhibitory effect of Sy6PGDH at 1 mM [17]. The absolute concentration (μmol/g-drycell weight) of Cit is approximately seven times that of 2OG and the second highest concentration among that of quantified organic acids in Synechocystis 6803 [30]. Under nitrogen-depleted conditions, when the level of Cit decreases, SyG6PDH and Sy6PGDH activity increase [40]. These suggest that among the three organic acids, Cit mainly acts an inhibitor of SyG6PDH and Sy6PGDH in vivo (Figure 6). Kinetic analyses of SyG6PDH in the presence of Cit demonstrate that Cit is an uncompetitive inhibitor for G6P and a mixed inhibitor for NADP+ of SyG6PDH (Table 1). Kinetic analyses of Sy6PGDH in the presence of Cit demonstrate that Cit is a mixed inhibitor for 6PG and is an uncompetitive inhibitor for NADP+ of Sy6PGDH (Table 1). In contrast with SyG6PDH and Sy6PGDH, both G6PDH and 6PGDH from heterotrophic bacterium, Corynebacterium glutamicum are not inhibited by Cit [14].

Model drawing of the biochemical inhibition of the OPP pathway enzymes by citrate in the oxidative TCA cycle in Synechocystis sp. PCC 6803.

Figure 6.
Model drawing of the biochemical inhibition of the OPP pathway enzymes by citrate in the oxidative TCA cycle in Synechocystis sp. PCC 6803.

6PGL: 6-phosphogluconolactone, Ru5P: ribulose-5-phosphate, F6P: fructose-6-phosphate, GAP: glyceraldehyde-3-phosphate, Ici: isocitrate.

Figure 6.
Model drawing of the biochemical inhibition of the OPP pathway enzymes by citrate in the oxidative TCA cycle in Synechocystis sp. PCC 6803.

6PGL: 6-phosphogluconolactone, Ru5P: ribulose-5-phosphate, F6P: fructose-6-phosphate, GAP: glyceraldehyde-3-phosphate, Ici: isocitrate.

In Synechocystis 6803, a shift from light to dark conditions results in the accumulation of metabolites in the OPP pathway [41]. However, in a mutant strain not having SyG6PDH, a shift from light to dark conditions results in the accumulation of metabolites in the oxidative TCA cycle rather than the OPP pathway [41]. Unlike nitrogen-fixing cyanobacteria [42], Synechocystis 6803 does not have the glyoxylate shunt [43] and Synechocystis 6803 generally runs the oxidative TCA cycle via the reactions catalyzed by isocitrate dehydrogenase (ICD) from Synechocystis 6803 (SyICD) and SySSADH (Figure 6). Although malate dehydrogenase from Synechocystis 6803 catalyzes the reaction generating NADH [44], SyICD [45] and SySSADH (Table 1) catalyze the reaction generating NADPH rather than NADH (Figure 6). Therefore, in addition to photosynthesis and the OPP pathway, the oxidative TCA cycle generates NADPH in Synechocystis 6803 (Figure 6). Also, Cit did not inhibit SyICD [45] and SySSADH (Figures 5 and 6). Thus, in Synechocystis 6803, the inhibition by Cit is specific to the dehydrogenases of the OPP pathway and not the oxidative TCA cycle (Figure 6). The Km value of SySSADH for SSA (0.010 mM) was higher than that of other bacterial SSADHs (three species: 0.003–0.0078 mM) [46–48] (Table 1). The Km value of SySSADH for NADP+ (0.058 mM) was within the range of that of bacterial SSADHs (four species: 0.0092–0.10 mM) [46–49] (Table 1). The kcat values of SySSADH (1.38 s−1 for SSA and 1.45 s−1 for NADP+) were markedly lower than those of other bacterial SSADHs (three species: 4.7–16.3 s−1) [47–49] (Table 1). A comparison of kinetic parameters among bacterial SSADHs revealed that the activity of SySSADH is markedly lower than that of other bacterial SSADHs. Similarly, the activity of other metabolic enzymes in the oxidative TCA cycle in Synechocystis 6803 is markedly lower than that of the same enzymes from other organisms [44,50,51]. Moreover, the catalytic efficiency [kcat/S0.5 (Km)] of SySSADH for NADP+ was markedly lower than that of two enzymes in the OPP pathway in Synechocystis 6803 (Table 1). These demonstrate that the oxidative TCA cycle is less active in generating NADPH than the OPP pathway in Synechocystis 6803 (Figure 6). This is corroborated by the previous reports that in contrast with the OPP pathway flux, the TCA cycle flux is low under dark heterotrophic and two kinds of photoheterotrophic conditions where NADPH generation via photosynthesis is repressed [6,8,52].

From the above, we presume that in order to avoid the overproduction of NADPH, the dehydrogenases of the OPP pathway (high-efficiency pathway in NADPH generation) are biochemically inhibited when Cit accumulate and the flow of the oxidative TCA cycle (low-efficiency pathway in NADPH generation) increases in Synechocystis 6803 (Figure 6). We demonstrated, for the first time, the biochemical relationship between the OPP pathway and the oxidative TCA cycle, which have been generally thought to produce different reducing powers, on the basis of different physiological significance. This finding contributed to the understanding of the regulation mechanism of sugar catabolism in cyanobacteria and made it easy to produce useful substances using cyanobacteria.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology, Japan, by a grant to T.O., from the JST-ALCA (Japan Science and Technology Agency, Advanced Low Carbon Technology Research and Development Program; Grant Number JPMJAL1306) and the Japan Society for the Promotion of Science, KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (Grant Number 16H06559).

Author Contributions

S.I. designed the research, performed the experiments, analyzed data and wrote the manuscript; T.O. wrote the manuscript.

Abbreviations

     
  • 2OG

    2-oxoglutarate

  •  
  • 6PG

    6-phosphogluconate

  •  
  • 6PGDH

    6-phosphogluconate dehydrogenase

  •  
  • 6PGL

    6-phosphogluconolactone

  •  
  • AcC

    acetyl-CoA

  •  
  • Arg

    l-arginine

  •  
  • Asp

    l-aspartate

  •  
  • Ca

    CaCl2

  •  
  • Cit

    citrate

  •  
  • F6P

    fructose-6-phosphate

  •  
  • FBP

    fructose-1,6-bisphosphate

  •  
  • Fum

    fumarate

  •  
  • G6P

    glucose-6-phosphate

  •  
  • G6PDH

    glucose-6-phosphate dehydrogenase

  •  
  • GAP

    glyceraldehyde-3-phosphate

  •  
  • Glc

    glucose

  •  
  • Gln

    l-glutamine

  •  
  • Glu

    l-glutamate

  •  
  • GST

    glutathione-S-transferase

  •  
  • ICD

    isocitrate dehydrogenase

  •  
  • Ici

    isocitrate

  •  
  • Mal

    l-malate

  •  
  • Mg

    MgCl2

  •  
  • OAA

    oxaloacetate

  •  
  • OPP pathway

    oxidative pentose phosphate pathway

  •  
  • PEP

    phosphoenolpyruvate

  •  
  • Pyr

    pyruvate

  •  
  • R5P

    ribose-5-phosphate

  •  
  • Ru5P

    ribulose-5-phosphate

  •  
  • SSA

    succinic semialdehyde

  •  
  • SSADH

    succinic semialdehyde dehydrogenase

  •  
  • Suc

    succinate

  •  
  • SyG6PDH

    G6PDH from Synechocystis 6803

  •  
  • SyICD

    ICD from Synechocystis 6803

  •  
  • Synechocystis 6803

    Synechocystis sp. PCC 6803

  •  
  • SySSADH

    SSADH from Synechocystis 6803

  •  
  • TCA cycle

    tricarboxylic acid cycle

References

References
1
Katayama
,
N.
,
Iijima
,
H.
and
Osanai
,
T.
(
2018
)
Production of bioplastic compounds by genetically manipulated and metabolic engineered cyanobacteria
.
Adv. Exp. Med. Biol.
1080
,
155
169
2
Oliver
,
N.J.
,
Rabinovitch-Deere
,
C.A.
,
Carroll
,
A.L.
,
Nozzi
,
N.E.
,
Case
,
A.E.
and
Atsumi
,
S.
(
2016
)
Cyanobacterial metabolic engineering for biofuel and chemical production
.
Curr. Opin. Chem. Biol.
35
,
43
50
3
Kaneko
,
T.
,
Sato
,
S.
,
Kotani
,
H.
,
Tanaka
,
A.
,
Asamizu
,
E.
,
Nakamura
,
Y.
et al (
1996
)
Sequence analysis of the genome of the unicellular cyanobacterium synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions
.
DNA Res.
3
,
109
136
4
Branco Dos Santos
,
F.
,
Du
,
W.
and
Hellingwerf
,
K.J.
(
2016
)
Synechocystis: not just a plug-bug for CO2, but a green E. coli
.
Front. Bioeng. Biotechnol.
4
,
32
5
Osanai
,
T.
,
Shirai
,
T.
,
Iijima
,
H.
,
Nakaya
,
Y.
,
Okamoto
,
M.
,
Kondo
,
A.
et al (
2015
)
Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium
.
Front. Microbiol.
6
,
1064
6
Zhang
,
S.
and
Bryant
,
D.A.
(
2011
)
The tricarboxylic acid cycle in cyanobacteria
.
Science
334
,
1551
1553
7
Chen
,
X.
,
Schreiber
,
K.
,
Appel
,
J.
,
Makowka
,
A.
,
Fähnrich
,
B.
,
Roettger
,
M.
et al (
2016
)
The Entner–Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants
.
Proc. Natl. Acad. Sci. U.S.A.
113
,
5441
5446
8
Wan
,
N.
,
DeLorenzo
,
D.M.
,
He
,
L.
,
You
,
L.
,
Immethun
,
C.M.
,
Wang
,
G.
et al (
2017
)
Cyanobacterial carbon metabolism: fluxome plasticity and oxygen dependence
.
Biotechnol. Bioeng.
114
,
1593
1602
9
Ueda
,
K.
,
Nakajima
,
T.
,
Yoshikawa
,
K.
,
Toya
,
Y.
,
Matsuda
,
F.
and
Shimizu
,
H.
(
2018
)
Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803
.
J. Biosci. Bioeng.
126
,
38
43
10
You
,
L.
,
He
,
L.
and
Tang
,
Y.J.
(
2015
)
Photoheterotrophic fluxome in Synechocystis sp. strain PCC 6803 and its implications for cyanobacterial bioenergetics
.
J. Bacteriol.
197
,
943
950
11
Osanai
,
T.
,
Numata
,
K.
,
Oikawa
,
A.
,
Kuwahara
,
A.
,
Iijima
,
H.
,
Doi
,
Y.
et al (
2013
)
Increased bioplastic production with an RNA polymerase sigma factor SigE during nitrogen starvation in Synechocystis sp. PCC 6803
.
DNA Res.
20
,
525
535
12
Choi
,
Y.N.
and
Park
,
J.M.
(
2016
)
Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803
.
Bioresour. Technol.
213
,
54
57
13
Lin
,
P.C.
,
Saha
,
R.
,
Zhang
,
F.
and
Pakrasi
,
H.B.
(
2017
)
Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocystis sp. PCC 6803
.
Sci. Rep.
7
,
17503
14
Moritz
,
B.
,
Striegel
,
K.
,
De Graaf
,
A.A.
and
Sahm
,
H.
(
2000
)
Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo
.
Eur. J. Biochem.
267
,
3442
3452
15
Kato
,
N.
,
Sahm
,
H.
,
Schütte
,
H.
and
Wagner
,
F.
(
1979
)
Purification and properties of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from a methanol-utilizing yeast, Candida boidinii
.
Biochim. Biophys. Acta
566
,
1
11
16
Hagen
,
K.D.
and
Meeks
,
J.C.
(
2001
)
The unique cyanobacterial protein OpcA is an allosteric effector of glucose-6-phosphate dehydrogenase in Nostoc punctiforme ATCC 29133
.
J. Biol. Chem.
276
,
11477
11486
17
Ito
,
S.
and
Osanai
,
T.
(
2018
)
Single amino acid change in 6-phosphogluconate dehydrogenase from Synechocystis conveys higher affinity for NADP+ and altered mode of inhibition by NADPH
.
Plant Cell Physiol.
59
,
2452
2461
18
Ito
,
S.
,
Takeya
,
M.
and
Osanai
,
T.
(
2017
)
Substrate specificity and allosteric regulation of a d-lactate dehydrogenase from a unicellular cyanobacterium are altered by an amino acid substitution
.
Sci. Rep.
7
,
15052
19
Dixon
,
M.
and
Webb
,
E.C.
(
1979
)
Enzymes
,
Longman
,
London
, pp.
400
402
20
Opheim
,
D.
and
Bernlohr
,
R.W.
(
1973
)
Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis
.
J. Bacteriol.
116
,
1150
1159
21
Hansen
,
T.
,
Schlichting
,
B.
and
Schönheit
,
P.
(
2002
)
Glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzyme
.
FEMS Microbiol. Lett.
216
,
249
253
22
Lessie
,
T.G.
and
Wyk
,
J.C.
(
1972
)
Multiple forms of Pseudomonas multivorans glucose-6-phosphate and 6-phosphogluconate dehydrogenases: differences in size, pyridine nucleotide specificity, and susceptibility to inhibition by adenosine 5'-triphosphate
.
J. Bacteriol.
110
,
1107
1117
23
Ben-Bassat
,
A.
and
Goldberg
,
I.
(
1980
)
Purification and properties of glucose-6-phosphate dehydrogenase (NADP+/NAD+) and 6-phosphogluconate dehydrogenase (NADP+/NAD+) from methanol-grown Pseudomonas C
.
Biochim. Biophys. Acta
611
,
1
10
24
TranNgoc
,
K.
,
Pham
,
N.
,
Lee
,
C.
and
Jang
,
S.H.
(
2019
)
Cloning, expression, and characterization of a psychrophilic glucose 6-phosphate dehydrogenase from Sphingomonas sp. PAMC 26621
.
Int. J. Mol. Sci.
20
,
E1362
25
McCarthy
,
J.K.
,
O'Brien
,
C.E.
and
Eveleigh
,
D.E.
(
2003
)
Thermostable continuous coupled assay for measuring glucose using glucokinase and glucose-6-phosphate dehydrogenase from the marine hyperthermophile Thermotoga maritima
.
Anal. Biochem.
318
,
196
203
26
Rauch
,
B.
,
Pahlke
,
J.
,
Schweiger
,
P.
and
Deppenmeier
,
U.
(
2010
)
Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans
.
Appl. Microbiol. Biotechnol.
88
,
711
718
27
Lee
,
W.T.
and
Levy
,
H.R.
(
1992
)
Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction
.
Protein Sci.
1
,
329
334
28
Banerjee
,
S.
and
Fraenkel
,
D.G.
(
1972
)
Glucose-6-phosphate dehydrogenase from Escherichia coli and from a “high-level” mutant
.
J. Bacteriol.
110
,
155
160
29
Özkul
,
K.
and
Karakaya
,
H.
(
2015
)
Characterisation of an opcA mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803
.
Curr. Microbiol.
71
,
572
578
30
Dempo
,
Y.
,
Ohta
,
E.
,
Nakayama
,
Y.
,
Bamba
,
T.
and
Fukusaki
,
E.
(
2014
)
Molar-based targeted metabolic profiling of cyanobacterial strains with potential for biological production
.
Metabolites
4
,
499
516
31
Acero-Navarro
,
K.E.
,
Jiménez-Ramírez
,
M.
,
Villalobos
,
M.A.
,
Vargas-Martínez
,
R.
,
Perales-Vela
,
H.V.
and
Velasco-García
,
R.
(
2018
)
Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa
.
Protein Expre. Purif.
142
,
53
61
32
Fuentealba
,
M.
,
Muñoz
,
R.
,
Maturana
,
P.
,
Krapp
,
A.
and
Cabrera
,
R.
(
2016
)
Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: simulation, kinetics and evolutionary studies
.
PLoS One
11
,
e0152403
33
Bonsignorea
,
A.
and
De Flora
,
A.
(
1972
)
Regulatory properties of glucose-6-phosphate dehydrogenase
.
Curr. Top. Cell. Reg.
6
,
21
62
34
Ishikawa
,
Y.
,
Miyagi
,
A.
,
Ishikawa
,
T.
,
Nagano
,
M.
,
Yamaguchi
,
M.
,
Hihara
,
Y.
et al (
2019
)
One of the NAD kinases, sll1415, is required for the glucose metabolism of Synechocystis sp. PCC 6803
.
Plant. J.
98
,
654
666
35
Saha
,
R.
,
Liu
,
D.
,
Hoynes-O'Connor
,
A.
,
Liberton
,
M.
,
Yu
,
J.
,
Bhattacharyya-Pakrasi
,
M.
et al (
2016
)
Diurnal regulation of cellular processes in the cyanobacterium Synechocystis sp. strain PCC 6803: insights from transcriptomic, fluxomic, and physiological analyses
.
mBio
7
,
e00464-16
36
Young
,
J.D.
,
Shastri
,
A.A.
,
Stephanopoulos
,
G.
and
Morgan
,
J.A.
(
2011
)
Mapping photoautotrophic metabolism with isotopically nonstationary 13C flux analysis
.
Metab. Eng.
13
,
656
665
37
Levy
,
H.R.
(
1979
)
Glucose-6-phosphate dehydrogenases
.
Adv. Enzymol. Relat. Areas Mol. Biol.
48
,
97
192
38
Werner
,
A.
,
Broeckling
,
C.D.
,
Prasad
,
A.
and
Peebles
,
C.A.M.
(
2019
)
A comprehensive time-course metabolite profiling of the model cyanobacterium Synechocystis sp. PCC 6803 under diurnal light:dark cycles
.
Plant. J.
99
,
379
388
39
Krebs
,
H.A.
(
1942
)
The effect of inorganic salts on the ketone decomposition of oxaloacetic acid
.
Biochem. J.
36
,
303
305
40
Osanai
,
T.
,
Oikawa
,
A.
,
Shirai
,
T.
,
Kuwahara
,
A.
,
Iijima
,
H.
,
Tanaka
,
K.
et al (
2014
)
Capillary electrophoresis-mass spectrometry reveals the distribution of carbon metabolites during nitrogen starvation in Synechocystis sp. PCC 6803
.
Environ. Microbiol.
16
,
512
524
41
Maruyama
,
M.
,
Nishiguchi
,
H.
,
Toyoshima
,
M.
,
Okahashi
,
N.
,
Matsuda
,
F.
and
Shimizu
,
H.
(
2019
)
Time-resolved analysis of short term metabolic adaptation at dark transition in Synechocystis sp. PCC 6803
.
J. Biosci. Bioeng.
128
,
424
428
42
Zhang
,
S.
and
Bryant
,
D.A.
(
2015
)
Biochemical validation of the glyoxylate cycle in the cyanobacterium Chlorogloeopsis fritschii strain PCC 9212
.
J. Biol. Chem.
290
,
14019
14030
43
Knoop
,
H.
,
Gründel
,
M.
,
Zilliges
,
Y.
,
Lehmann
,
R.
,
Hoffmann
,
S.
,
Lockau
,
W.
et al (
2013
)
Flux balance analysis of cyanobacterial metabolism: the metabolic network of Synechocystis sp. PCC 6803
.
PLoS Comput. Biol.
9
,
e1003081
44
Takeya
,
M.
,
Ito
,
S.
,
Sukigara
,
H.
and
Osanai
,
T.
(
2018
)
Purification and characterisation of malate dehydrogenase from Synechocystis sp. PCC 6803: Biochemical barrier of the oxidative tricarboxylic acid cycle
.
Front. Plant. Sci.
9
,
947
45
Muro-Pastor
,
M.I.
and
Florencio
,
F.J.
(
1992
)
Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803
.
Eur. J. Biochem.
203
,
99
105
46
Jaeger
,
M.
,
Rothacker
,
B.
and
Ilg
,
T.
(
2008
)
Saturation transfer difference NMR studies on substrates and inhibitors of succinic semialdehyde dehydrogenases
.
Biochem. Biophys. Res. Commun.
372
,
400
406
47
Wang
,
X.
,
Lai
,
C.
,
Lei
,
G.
,
Wang
,
F.
,
Long
,
H.
,
Wu
,
X.
et al (
2018
)
Kinetic characterization and structural modeling of an NADP+-dependent succinic semialdehyde dehydrogenase from Anabaena sp. PCC7120
.
Int. J. Biol. Macromol.
108
,
615
624
48
De Carvalho
,
L.P.
,
Ling
,
Y.
,
Shenm
,
C.
,
Warren
,
J.D.
and
Rhee
,
K.Y.
(
2011
)
On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis
.
Arch. Biochem. Biophys.
509
,
90
99
49
Jang
,
E.H.
,
Park
,
S.A.
,
Chi
,
Y.M.
and
Lee
,
K.S.
(
2014
)
Kinetic and structural characterization for cofactor preference of succinic semialdehyde dehydrogenase from Streptococcus pyogenes
.
Mol. Cells
37
,
719
726
50
Katayama
,
N.
,
Takeya
,
M.
and
Osanai
,
T.
(
2019
)
Biochemical characterisation of fumarase C from a unicellular cyanobacterium demonstrating its substrate affinity, altered by an amino acid substitution
.
Sci. Rep.
9
,
10629
51
Ito
,
S.
,
Koyama
,
N.
and
Osanai
,
T.
(
2019
)
Citrate synthase from Synechocystis is a distinct class of bacterial citrate synthase
.
Sci. Rep.
9
,
6038
52
Nakajima
,
T.
,
Kajihata
,
S.
,
Yoshikawa
,
K.
,
Matsuda
,
F.
,
Furusawa
,
C.
,
Hirasawa
,
T
et al et al (
2014
)
Integrated metabolic flux and omics analysis of Synechocystis sp. PCC 6803 under mixotrophic and photoheterotrophic conditions.
Plant Cell Physiol.
55
,
1605
1612
.