Enzyme mechanism analysis of UDP-glucose pyrophosphorylase by X-ray crystal analysis

Research Project

Project/Area Number	09680591
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Structural biochemistry
Research Institution	Osaka University
Principal Investigator	KUSUNOKI Masami Institute for Protein Research, Osaka University, Associate Professor, 蛋白質研究所, 助教授 (90135749)
Co-Investigator(Kenkyū-buntansha)	TANIZAWA Katsuyuki Institute of Scientific and Industrial Research Osaka University, Professor, 産業科学研究所, 教授 (20133134)
Project Period (FY)	1997 – 1998
Project Status	Completed (Fiscal Year 1998)
Budget Amount *help	¥2,900,000 (Direct Cost: ¥2,900,000) Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000) Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
Keywords	Enzyme / X-ray Analysis / UDP / Crystal / 反応機構
Research Abstract	UDP-glucose pyrophosphorylase catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose-1-phosphate and MgUTP.We isolated and purified cDNA encoding UDP-glucose pyrophosphorylase from potato tuber.It has 477 amino acid residues.The enzyme was crystallized by the hanging-drop vapor diffusion method with a precipitant of ammonium sulfate.The space group is P212121 with cell dimensions a=108.2, b=124.7, c=87.1 and VM=2.8 /dalton.The crystal structure was solved by multiple-isomorphous replacement with four heavy atom derivatives, K2Pt(CN)4, Hg(CH3COO)2, UO2(NO3)2 and Sm2(SO4)3.The intensity data were collected by an imaging plate diffractometer, RIGALU RAXIS IIc. The average figure of merit at 3.0 resolution is 0.43, using "mlphare" in CCP4 program package.The phase improvement was carried out by "dm" in CCP4, resulting in a 2.2 resolution density map.The density modification includes histogram-mapping, constraint of Sayer formula, non-crystallographic averaging and solvent flattening, with assumption of 41% solvent content.The final free-R-value was 0.27 at 2.2 resolution. Two identical molecules are in the crystallographic asymmetric unit.The two molecules are related by 143 degree rotation around an axis almost parallel to the crystallographic z axis with some translation.Each molecule consists of two domains.The N-terminal domain has five helices and seven b-strands (a/b structure) with two additional long helices in N-terminus.The C-terminal domain is mainly composed of left-handed b-helix similar to the structure of UDP-N-acetylglucosamine acyltransferase.The protein structure has been refined by the program X-PLOR.