Sedoheptulose 1,7-bisphosphatase (glpX) from Bacillus methanolicus
Usage and Biology
The SBPase is one of enzymes needed for the Calvin cycle. It catalyzes the reaction from sedoheptulose 1,7-bisphosphate to sedoheptulose 7-phosphate as shown in Figure 1. The enzyme is characteristic for the part of sedoheptulose 7-phosphate regeneration in the Calvin-cycle. It was shown before that oveerexpression of the SBPase in tobacco results in enhanced carbon assimilation and crop yield (Rosenthal et al., 2011). SBPases are homodimers with two identical subunits of 35kD to 38kD. The KM-value of the SBPase homologue GlpX of Bacillus methanolicus is 14 ± 0.5 µM (Stolzenberger et al., 2013).
E. coli does not have a SBPase homologue which is needed E. coli for enabling the whole cycle.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
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 10: Approaches by adding one enzyme at each step. Each step shows the activity of the enzymes.
- 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).
Rosenthal et al., 2011. Overexpressing the C(3) photosynthesis cycle enzyme sedoheptulose 1,7-bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO(2) fumigation (FACE). BMC Plant Biol., vol. 11, pp. 123