Development of a Novel Assay for Synthesis and Hydrolysis of Sedoheptulose 1,7-bisphosphate (SBP) in vitro by Combinations of Purified Fructose 1,6-bisphosphate aldolases (FBA) Proteins and Fructose 1,6-bisphosphatases (FBPase) Proteins from Bacillus methanolicus MGA3

Materials and Reagents

1. Transketolase (TKT) from S. cerevisiae (Sigma-Aldrich)
   
2. Purified proteins: Fructose 1,6-bisphosphatase (GlpX^C) and promiscuous fructose 1,6-bisphosphatase/Sedoheptulose 1,7-bisphosphatase (GlpX^P), as well as promiscuous fructose 1,6-bisphosphate aldolase/ Sedoheptulose 1,7-bisphosphate aldolase (FBA^C and FBA^P) from B. methanolicus MGA3
   
3. Sephadex G25 gel filtration (Amersham Biosciences )
   
4. Factor Xa (Novagen)
   
5. *(1 M) 50 mM Tris-HCl (pH 7.5)
   
6. *(400 mM) 20 mM D-fructose 6-phosphate dipotassium salt (F6-P) (Sigma-Aldrich)
   
7. *(400 mM) 20 mM DL-glyceraldehyd 3-phosphate solution (GAP) (Sigma-Aldrich)
   
8. *(400 mM) 20 mM dihydroxyacetone phosphate dilithium salt (DHAP)
   
9. *(200 mM) 10 µM thiamine pyrophosphate (TPP) (Sigma-Aldrich)
   
10. *(40 mM) 2 mM MnCl₂
    
11. *(200 mM) 10 mM ammonium bicarbonate solution (pH 9.3)
    Note: *Concentration of stock solution are given in brackets.
    

Equipment

1. Amicon Ultra-0.5 centrifugal filter (Millipore)
   
2. LaChromUltra HPLC system (Hitachi)
   
3. MicroTOF-Q hybrid quadrupole/time-of-flight mass spectrometer (BD)
   
4. SeQuant ZIC-pHILLIC column (150 x 2.1 mm) (Merck KGaA)
   

Software

1. Compass software 1.3 (BD)

Procedure

1. Enzyme assays for the purified GlpX proteins in vitro
   1. The FBA activity in the direction of SBP synthesis was done by using a discontinuous, coupled enzyme assay containing TKT (from S. cerevisiae), recombinant GlpX^C and GlpX^P, as well as FBA^C and FBA^P from B. methanolicus as described below.
      
   2. Because E4-P is not acquired by purchase, E4-P was generated in a pre-reaction by the TKT (5 U/mg) from F6-P and GAP.
      
   3. Protein production and purification was done as follows:
      
      1. The enzymes were cloned into pET16b, which allows IPTG-inducible expression with an N-terminal decahistidyl tag and a factor Xa cleavage site in Escherichia coli (E. coli) BL21 (DE3).
      2.  The induction with 0.5 mM IPTG was started at an OD₆₀₀ of 0.5 to 0.6 in LB. The cells (500 ml) were harvested 4 h after induction and washed in 20 mM Tris, 300 mM NaCl, 5 mM imidazol, and 5% (vol/vol) glycerol.
      3.  Pelleted cells were stored at 20 °C until protein purification.
      4. Prior to lysis by French press, cells were resuspended in 20 mM Tris, 300 mM NaCl, 5 mM imidazol, and 5% (vol/vol) glycerol, and protease activity was inhibited by addition of 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM diisopropylfluorophosphate (DFP).
      5. The extract was cleared by centrifugation for 1.5 h at 140,000 x g.
      6. Peak fractions of Ni-nitrilotriacetic acid (Ni-NTA) agarose affinity chromatography eluted with 20 mM Tris, 300 mM NaCl, 100, 200, or 400 mM imidazol, and 5% (vol/vol) glycerol were pooled, and the pooled fractions were desalted using Sephadex G25 gel filtration and buffered in 50 mM Tris/HCl (pH 7.5).
      7. After purification, the His tag was cleaved by factor Xa according to the manufacturer’s recommendations.
         
   4. The purified protein was buffered in 50 mM Tris-HCl (pH 7.5). The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 20 mM F6-P, 20 mM GAP, 20 mM DHAP, 10 µM thiamine pyrophosphate (TPP), 2 mM MnCl₂ and 3 U/mg of each purified enzyme (FBA^C or FBA^P and GlpX^C or GlpX^P).
      
   5. The reaction was started by addition of TKT (5 U/mg). The detection of the generated products was performed via liquid chromatography–mass spectrometry (LC-MS) as described below.
      
   6. The assay was performed at 50 °C for 45 min in volume of 1 ml. The reaction was stopped by purification of the containing enzymes using Amicon Ultra-0.5 centrifugal filter. Therefore, the reaction mixture was sampled to the Amicon^® Ultra filter device and cap it and the device was centrifuged at 14,000 x g for approximately 30 min. Thereby the proteins were filtered out of the mixture but the remaining incredients passed. The supernatant was then free of protein and the incredients were analyzed by LCMS. The expectations and results and described in a separate section below.
   
   
2. LC-MS analysis of the products after enzyme assay
   1. LC-MS data were obtained using a LaChromUltra HPLC system coupled to a microTOF-Q hybrid quadrupole/time-of-flight mass spectrometer.
      
   2. For Ionization the mass spectrometer is equipped with an electrospray ionization (ESI) source. Separation of the samples via HPLC was carried out by a SeQuant ZIC-pHILLIC column using the solvents 10 mM ammonium bicarbonate solution (pH 9.3) as eluent A and acetonitrile as eluent B.
      
   3. The injection volume was 2 µl, flow rate was set to 150 µl min^-1, and gradient elution was performed as follows: t = 0 min, 80% B; t = 30 min, 10% B; t = 35 min, 10% B; t = 40 min 80% B; t = 60 min 80% B.
      
   4. MS detection was performed via ESI source in negative ionization mode. Nitrogen was applied as sheath, dry and collision gas.
      
   5. For internal mass calibration a solution of formate (0.1 M) in 50 % (v/v) isopropanol was injected within each MS analysis.
      
   6. MSMS analyses were performed by the auto MSMS mode of the microTOF-Q.
      
      Table 1. Parameters for microTOFcontrol in full scan MS mode
      

      Mode	Scan Mode Mass Range Spectra Acquisition Include Profile Spectra Focus	MS 50-1000 m/z Save Spectra Always Inactive	Ion Polarity Rolling Average Absolute Threshold Peak Summation Width Acquisition Rate	Positive off 10 5 pts 1.0 Hz
      Source	Endplate Offset Capillary Nebulizer	-500 V + 4500 V 2.0 bar	Dry Gas Dry Temp	8.0 L / min 180 °C
      Transfer	Funnel 1 RF Funnel 2 RF	300.0 Vpp 300.0 Vpp	ISCID Energy Hexapole RF	0.0 eV 200.0 Vpp
      Quadrupole	Ion Energy	4.0 eV	Low Mass	150.00 m/z
      Collision Cell	Collision Energy Transfer Time	8.0 eV 75.0 µs	Collision RF Pre Puls Storage	130.0 Vpp 5.0 µs

      
      
   7. Raw data were analyzed using the Compass software 1.3. Automatic internal mass calibration was achieved using the HPC quadratic algorithm.
      
   8. Identification of compounds was performed either by the specific mass to charge ratio and the retention time or by comparing the fragment ions in MSMS mode.
      
      Table 2. Additional parameters for microTOFcontrol in MS/MS (MRM) mode
      

      Auto MSMS	Precursor Ions	3	Threshold	6000 counts
      Transfer	Funnel 1 RF Funnel 2 RF	300.0 Vpp 300.0 Vpp	ISCID EnergyHexapole RF	0.0 eV 200.0 Vpp
      Quadrupole	Ion Energy	4.0 eV	Low Mass	150.00 m/z
      Collision Cell	Collision Energy Transfer Time	8.0 eV 75.0 µs	Collision RF Pre Puls Storage	130.0 Vpp 5.0 µs

      
      

Notes about results/experimental setup

1. The assay was performed to detect the synthesis and hydrolysis of SBP in vitro by combinations of purified FBA proteins and FBPase proteins from B. methanolicus MGA3. In the SBPase variant of the RuMP cycle, SBP is produced from E4-P and DHAP by SBA and dephosphorylated to yield S7-P by SBPase. Unfortunately, since neither E4-P nor SBP is commercially available, these compounds cannot be used directly in enzyme assays to obtain evidence for synthesis and hydrolysis of SBP. To circumvent this limitation, a coupled discontinuous enzyme assay including transketolase from S. cerevisiae was used.
2. Various combinations of substrates and enzymes were tested (Figure 1). No evidence for instabilities of the sugar phosphates at 50 °C was obtained when the substrates were incubated without enzymes.
   
   
   Figure 1. Determination of the sugar phosphate intermediates of the RuMP cycle using liquid chromatography-mass spectrometry (LC-MS). Shown is the experimental setup and determination of the sugar phosphate intermediates of the RuMP cycle using liquid chromatography-mass spectrometry (LC-MS). For the identification of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate, MS/MS was used. (A) Scheme of the substrate and enzyme combinations used in the assay. X indicates presence in the assay. (B) Presence (check marks) or absence of the indicated sugar phosphates, as detected by LC-MS/MS analysis. (C) LC-MS spectra of FBP and SBP.
   
   
3. E4-P and xylulose 5-phosphate (XU5-P) were generated from F-6P and GAP by transketolase from S. cerevisiae (Figure 1, line 1). Aldol condensation of E-4P with DHAP to yield SBP could be detected for both purified FBA^C or FBA^P from B. methanolicus (Figure 1, line 2). LC-MS/MS analysis confirmed the identity of SBP, which showed the expected mass shift of 30 Da (-CHOH-) compared to FBP (Figure 1, C). Thus, these results indicated that both FBAs from B. methanolicus are active as SBAs in vitro. Subsequently, hydrolysis of SBP to S7-P was assayed by using purified GlpX^C or GlpX^P from B. methanolicus. Hydrolysis of SBP and formation of S7-P occurred only when GlpX^P, but not GlpX^C, was added (Figure 1, line 3, 4). Thus, for the major FBPase of B. methanolicus, GlpX^C, evidence for SBPase activity could not be obtained in vitro, while GlpX^P showed activity as an SBPase.
   

Abbreviations

Enzymes
FBA     fructose-bisphosphate aldolase (EC 4.1.2.13)
TKT     transketolase (EC 2.2.1.1)
GlpX     fructose-bisphosphatase (EC 3.1.3.1)
SBA     sedoheptulose 1,7-bisphosphate aldolase (EC 4.1.2.x)
SBPase     sedoheptulose 1,7-bisphosphate (EC 3.1.3.37)

Metabolites
F6-P     fructose-6-phosphate
FBP     fructose-1,6-bisphosphate
GAP     glyceraldehyde 3-phosphate
DHAP     dihydroxyacetone phosphate
E4-P     erythrose 4-phosphate
SBP     sedoheptulose 1,7-bisphosphate
S7-P     sedoheptulose-7-phosphate
Ri5-P     ribose 5-phosphate
X5P     xylulose 5-phosphate
Ru5P     ribulose 5-phosphate

Acknowledgments

We would like to acknowledge Dr Trygve Brautaset for providing the strain B. methanolicus MGA3 and the plasmid pTH1; Dr Sonja Siwiora Brenke for providing the facilities, equipment and technical assistance to perform size exclusion chromatography and molecular mass estimation of the purified protein. This work was supported by SynMet, a 09-EuroSYNBIO-FP-023 project, funded in part by DFG through grant no. WE 2320/2-1.

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