Characterization

For the characterization of the sedoheptulose 1,7-bisphosphatase (SBPase / glpX) we did an enzyme assay with a His-Tag purification as described before (Stolzenberger et al., 2013).
The proteins were overexpressed by adding 1 mM IPTG for inducing the T7 promotor(BBa_K1465229). The increasing amount of protein could be verified with a SDS-PAGE. (Figure 2, 3 and 4).

Figure 2: Proteinexpression of Fba

Figure 3: Proteinexpression of Tkt

Figure 4: Proteinexpression of GlpX

All three SDS-Pages showed a band with a clear increase in the amount of protein like described in 'Stolzenberger et al., 2013'. We purified the transketolase (Tkt) and the fructose bisphosphate aldolase (Fba) as well as the sedoheptulose 1,7-bisphosphatase with the His-Tag mediated purification system (Figure 5, 6 and 7).

Figure 5: Protein purification of Fba

Figure 6: Protein purification of Tkt

Figure 7: Protein purification of GlpX

For the purified enzymes we performed a Bradford assay.

Figure 8: Bradford assay with purified enzymes (two technical and two biological replicates)

The Bradford assay (Figure 8) showed high concentrations of Tkt and Fba as well as a very low concentration of GlpX. After the purification we performed an enzyme assay as shown below (Figure 9).

Figure 9: Schematical view of the SBPase assay

Molecule Formula Molecular weight [M-H] Fructose-6-P C6H15O9P 259.021 Glyceraldehyd-3-P C3H7O6P 168.99 Dihydroxyacetone-P C3H7O6P 168.99 Xylulose-5-P C5H11O8P 229.01 Erythrose-4-P C4H9O7P 199.00 Sedoheptulose-7-P C7H15O10P 289.03 Sedoheptulose-1,7-BP C7H16O15P2 368.99

The product of the reaction, sedoheptulose 7-phosphate, could be identified via HPLC. We made different approaches for the enzyme assay to characterize all reactions (Figure 10).



Reaction mix:

• 20 mM Fructose 6-phosphate
• 20 mM Glyceraldehyde 3-phosphate
• 20 mM Dihydroxyacetonephosphate
• 10 µM Thiamine pyrophosphate
• 2 mM Manganese chloride
• 50 mM Tris-HCl

For the first approach we added no enzyme to verify that no product is generated as shown in Figure 11. The second approach includes the transketolase which catalyzes the reaction of F6P and GAP to erythrose 4-phosphate. For the third approach fructose bisphosphate aldolase was added to convert erythrose 4-phosphate with dihydroacetonephosphate to sedoheptulose 1,7-bisphosphate. For the last approach the sedoheptulose 1,7-bisphosphatase (GlpX) was added which results in sedoheptulose 7-phosphate. All intermediates could be verified in all approaches as expected. This measurement showed the activity of the SBPase in vitro (Xylulose 5-phosphate is a byproduct of one of the enzymatical reactions).

Figure 11: Comparison of 37°C and 50°C of the in vitro assay (all enzymes).

We did a comparison between 37°C and 50°C of the in vitro assay to investigate the different enzymatic activities as shown in Figure 11. The transketolase and aldolase showed a higher activity at 37°C which resulted in more respectively products. The sedoheptulose 1,7-bisphosphatase activity is higher at 50°C but also shows activity at 37°C. We could identify this characteristic because of the accumulation of sedoheptulose 1,7-bisphosphate in the 37°C approach. The second approach has a lower concentration of this sedoheptulose 1,7-bisphosphate but showed a higher concentration of sedoheptulose 7-phosphate which is due to the higher activity of the SBPase at 50°C. This results suggest that it is possible to enable SBPase activity with GlpX in E. coli.