Tesmer J J, Stemmler T L, Penner-Hahn J E, Davisson V J, Smith J L
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Proteins. 1994 Apr;18(4):394-403. doi: 10.1002/prot.340180410.
We have initiated a project to determine the three-dimensional structure of GMP synthetase (GMPS) from Escherichia coli. GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the glutamine amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics. The E. coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified. Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl2, ATP, and XMP. The crystals are monoclinic, space group P2(1), with cell parameters of a = 156.0 A, b = 102.0 A, c = 78.8 A, beta = 96.7 degrees. Diffraction data to 2.8 A spacings were collected on a Xuong-Hamlin area detector with an overall Rsym of 5.2%. Both the volume of the unit cell and the peaks in the self-rotation function are consistent with one GMPS tetramer of D2 symmetry in the crystallographic asymmetric unit. Previously, GMPS has been observed only as a dimer in solution. GMPS was covalently modified with p-chloromercuribenzylsulfonic acid (PCMBS), and its X-ray fluorescence spectrum was measured through the LIII absorption edge of mercury. Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi-wavelength anomalous diffraction was evaluated. The optimal MAD dispersive signal is 4.5% of magnitude of F, and the optimal MAD Bijvoet signal is 7.5% of magnitude of F at a concentration of approximately 1 mercury per 10-kDa protein. The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins.
我们启动了一个项目，旨在确定大肠杆菌中鸟苷酸合成酶（GMPS）的三维结构。GMPS在鸟嘌呤核苷酸从头生物合成的最后一步催化XMP转化为GMP，是谷氨酰胺酰胺转移酶家族的一员：这是一组负责将氮同化到氨基酸、嘌呤和嘧啶碱基、氨基糖及抗生素等化合物中的酶。编码GMPS的大肠杆菌guaA基因被克隆到一个tac表达载体中，进行过量表达，并对其基因产物进行纯化。利用重组GMPS在MgCl2、ATP和XMP存在的条件下，摸索出了蛋白质晶体生长的条件。这些晶体为单斜晶系，空间群为P2(1)，晶胞参数为a = 156.0 Å，b = 102.0 Å，c = 78.8 Å，β = 96.7°。在一台Xuong-Hamlin面积探测器上收集了间距为2.8 Å的衍射数据，整体Rsym为5.2%。晶胞体积和自身旋转函数中的峰均与晶体学不对称单元中一个具有D2对称性的GMPS四聚体一致。此前，GMPS在溶液中仅观察到为二聚体。GMPS用对氯汞苄磺酸（PCMBS）进行共价修饰，并通过汞的LIII吸收边测量其X射线荧光光谱。从该光谱中得出了半胱氨酸汞的反常散射因子，并评估了通过多波长反常衍射确定结构的可行性。在每10 kDa蛋白质约含1个汞原子的浓度下，最佳MAD色散信号为F值大小的4.5%，最佳MAD Bijvoet信号为F值大小的7.5%。此处列出的反常散射因子应可转移至其他蛋白质中的半胱氨酸汞。
