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

从马铃薯块茎中分离纯化了编码UDP-葡萄糖焦磷酸化酶的cDNA477个氨基酸残基。以硫酸铵为沉淀剂，采用悬滴气相扩散法结晶了该酶。空间群为P212121，晶胞尺寸为a=108.2，b=124.7，c=87.1，Vm=2.8/dalton。用K_2PT(CN)4，Hg(CH3COO)_2四个重原子衍生物进行多重同象置换，确定了其晶体结构。UO_2(NO_3)_2和Sm_2(SO_4)_3的强度数据由成像板衍射仪RIGALU RAXIS IIc采集。在3.0分辨率下的平均优值系数为0.43，使用CCP4程序包中的“mlphare”进行位相改进，得到2.2分辨率密度图。密度修改包括直方图绘制、Sayer公式约束、非晶体平均化和溶剂平坦化，在溶剂含量为41%的假设下，最终的自由-R值为0.27，在2.2分辨率下，两个相同的分子在晶体不对称单元中。两个分子绕着几乎平行于晶体z轴的轴旋转143度，并有一些平移。每个分子由两个结构域组成。N-末端结构域有5个螺旋和7个b链(a/b结构)，N-末端有两个长螺旋。C-末端结构域主要由类似UDP-N-乙酰氨基葡萄糖酰基转移酶结构的左手b-螺旋组成用X-PLOR程序对蛋白质结构进行了优化。

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.