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

udp -葡萄糖焦磷酸化酶催化从udp -葡萄糖到MgPPi的可逆尿苷基转移，形成葡萄糖-1-磷酸和MgUTP。从马铃薯块茎中分离纯化了编码udp -葡萄糖焦磷酸化酶的cDNA。它有477个氨基酸残基。以硫酸铵为沉淀剂，采用悬垂蒸汽扩散法对酶进行结晶。空间组为P212121，单元尺寸a=108.2, b=124.7, c=87.1， VM=2.8 /道尔顿。用四种重原子衍生物K2Pt(CN)4、Hg(CH3COO)2、UO2(NO3)2和Sm2(SO4)3进行多同构置换，求解了晶体结构。强度数据由成像板衍射仪RIGALU RAXIS IIc采集。使用CCP4程序包中的“mlphare”，3.0分辨率下的平均优值为0.43。在CCP4中通过“dm”进行相位改进，得到了2.2分辨率的密度图。密度修正包括直方图映射、Sayer公式约束、非结晶平均和溶剂平坦化，假设溶剂含量为41%。在2.2分辨率下，最终的自由r值为0.27。两个相同的分子在晶体不对称单元中。这两个分子围绕一个几乎平行于晶体学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端结构域有5条螺旋和7条b链（a/b结构），在n端有两个额外的长螺旋。c端结构域主要由左旋b-螺旋组成，类似于udp - n -乙酰氨基葡萄糖酰基转移酶的结构。蛋白质结构已被X-PLOR程序改进。

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.