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[Abstract] Bacillus methanolicus (B. methanolicus) is a Gram-positive, thermotolerant, and facultative methylotrophic bacterium that can use the one-carbon (C1) compound methanol as a source of carbon and energy (Schendel et al., 1990; Arfman et al., 1997; Arfman et al., 1992). B. methanolicus uses the Ribulose monosphosphate (RuMP) cycle for the fixation of formaldehyde (Anthony, 1986; Brautaset et al., 2007). In the RuMP cycle, Sedoheptulose 1,7-bisphoasphate (SBP) can be produced from erythrose 4-phosohate (E4-P) and dihydroxyacetone phosphate (DHAP) by sedoheptulose 1,7-bisphosphate aldolase (SBA) and dephosphorylated to yield sedoheptulose 7-phosphate (S7-P) by sedoheptulose 1,7-bisphosphatase (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 Saccharomyces cerevisiae (S. cerevisiae) was used. E4-P and xylulose 5-phosphate (XU5-P) were generated from fructose 6-phosphate (F-6P) and glyceraldehyde 3-phosphate (GAP) by transketolase from S. cerevisiae. Aldol condensation of E-4P with DHAP to yield SBP was tested by using purified fructose 1,6-bisphosphate aldolase (FBA C or FBA P ) from B. methanolicus (Stolzenberger et al., 2013a). Subsequently, hydrolysis of SBP to S7-P was assayed by using purified fructose 1,6-bisphosphatase (GlpX C or GlpX P ) from B. methanolicus (Aldolases and phosphatases are not commercially available) (Stolzenberger et al., 2013b).

bio-protocol

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