LEFT-HANDED [[beta]]-HELIX PROTEIN UDP-GLUCOSE PYROPHOSPHORYLASE. Masami Kusunoki, Yasuyuki Kitagawa, Hisashi Naitou, Yukiteru Katsube, Yukiyo Sakamoto*, Katsuyuki Tanizawa* and Toshio Fukui*, Institute for Protein Research, Osaka University, Suita Osaka 565 JAPAN *The Institute of Scientific and Industrial Research, Osaka University, Ibaraki Osaka 567 JAPAN
UDP-glucose pyrophosphorylase catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose-l-phosphate and MgUTP. We isolated and purified cDNA encoding UDP-glucose pyrophosphorylase from potato tuber. It has 477 amino acid residues and no apparent sequence homology to other proteins. The enzyme was crystallized by the hanging-drop vapor diffusion method with the precipitant ammonium sulfate. The space group is P212121 with cell dimensions a=108.2, b=124.7, c=87.1 Å, VM=2.8 Å3/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 three-dimensional intensity data were collected by imaging plate diffractometer of RIGAKU RAXIS IIc. The average figure of merit at 3.0 Å 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 Å 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 Å 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 [[beta]]-strands ([[alpha]]/[[beta]] structure) with two additional long helices in N-terminus. The C-terminal domain is mainly composed of left-handed [[beta]]-helix similar to the structure of UDP-N-acetylglucosamine acyltransferase1). The protein structure is under refinement with program X-PLOR.
1) C. R. H. Raetz and S. L. Roderick, Science, 270, 997-1000(1995)