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

UDP-葡萄糖焦磷酸化酶（UDP-glucose pyrophosphorylase，UDP）催化尿苷酰从UDP-葡萄糖到MgPPi的可逆转移，形成葡萄糖-1-磷酸和MgUTP。我们从马铃薯块茎中分离纯化了UDP-葡萄糖焦磷酸化酶的cDNA。它含有477个氨基酸残基。该酶用硫酸铵沉淀的悬滴气相扩散法结晶。空间群为P212121，细胞尺寸a=108.2，B=124.7，c=87.1，VM=2.8 /道尔顿。晶体结构用四种重原子衍生物K_2 Pt（CN）_4，Hg（CH_3COO）_2，UO_2（NO_3）_2和Sm_2（SO_4）_3进行了多晶型置换。强度数据用成像板衍射仪RIGALU RAXIS IIc收集，3.0分辨率下的平均优值为0.43，用CCP 4程序包中的“mlphare”进行相位修正，用CCP 4程序包中的“dm”进行相位修正，得到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、非晶体学平均和溶剂平坦化，假设溶剂含量为41%。最终的游离-R-值为0.27，分辨率为2.2。两个相同的分子在晶体学不对称单元中。这两个分子通过围绕几乎平行于每个分子由两个结构域组成，N端结构域有5个螺旋和7个b-链（a/B结构），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-乙酰葡萄糖胺酰基转移酶的结构。

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.