Structure of a Dual Activity FBP/SBPase from Synechococcus PCC 7942

Research Project

Project/Area Number	13660098
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	応用微生物学・応用生物化学
Research Institution	Osaka Prefecture University
Principal Investigator	NISHIMURA Keiichiro Osaka Prefecture University, Research Institute for Advanced Science and Technology, Professor, 先端科学研究所, 教授 (70026558)
Co-Investigator(Kenkyū-buntansha)	TADA Toshiji Osaka Prefecture University, Research Institute for Advanced Science and Technology, Associate Professor, 先端科学研究所, 助教授 (70275288)
Project Period (FY)	2001 – 2003
Project Status	Completed (Fiscal Year 2003)
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)
Keywords	Cyanobacteria / Calvin cycle / X-Ray structure analysis / FBPase / SBPase / FBP / Allosteric regulation / Dual activity / 放射光 / GAPDH / FB Pace / SB Pace / 結晶化 / 異常分散 / 変異体 / セレノメチオニン
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 … More 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