| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Phosphate hydrolysis reactions in the Calvin cycle of higher plants are catalyzed respectively, by fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase). Crystallographic studies of FBPases have been widely conducted. However crystal structures of SBPases have not been reported. In Synechococcus PCC 7942 cells, the major isozyme of FBPase has been found to possess both FBPase and SBPase activities in vitro. So we named this enzyme fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase). When the enzyme was treated with a mixture of fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, the hydrolytic activity was higher than that with each of the substrates. The aim of this study was to clarify the molecular mechanisms of the novel enzyme, FBP/SBPase using its crystallographic structure.
FBP/SBPase of Synechococcus PCC 7942 was overexpressed by E.coli BL21(DE3)pLysS and purified by column chromatography. The protein used for crystallization was at
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10 mg ml^<-1> in 50 mM potassium phosphate buffer pH 8.0. High-quality crystals grew in the presence of AMP, which is an inhibitor of FBP/SBPase, using ammonium sulfate as a precipitant. A complete data set was collected on an R-AXIS IV imaging plate detector using synchrotron radiation of wavelength 0.7 Å at BL41XU station of SPring-8. The crystals diffracted to at least 2.2 Å resolution. The crystal system was determined to be monoclinic, P2_1, with unit-cell parameters a = 80.1, b = 84.2, c = 104.3 Å, b = 101.7°. Se-Met labeling of FBP/SBPase was done using the Met auxotroph of E. coli B834(DE3)pLysS. The purification and crystallization of the Se-Met FBP/SBPase were carried out using the same conditions as for the wild type. Multiwavelength diffraction data for MAD phasing of the Set-Met FBP/SBPase were collected at BL5B of PF.
The structure of FBP/SBPase was solved using MAD scattering by Se-Met residues. The enzyme has a molecular mass of 160 kDa and consists of four identical subunits, similar to typical FBPases. The topology of the subunit of FBP/SBPase, in which α-helices and β-sheet are stacked alternately, is similar to that of typical FBPase. However, the three-dimensional structure of the subunit and the quaternary structure of FBP/SBPase are quite different from those of FBPases. The subunit consists of two domains. AMP has been found to be located between two domains. The binding site and mode are different from those in the typical FBPase. The relatively large basin surrounded by acidic amino acids is estimated to be the substrate binding site. Less
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