Huang K, Kodandapani R, Kallwass H, Hogan J K, Parris W, Friesen J D, Gold M, Jones J B, James M N
Medical Research Council of Canada Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton.
Proteins. 1992 Apr;13(2):158-61. doi: 10.1002/prot.340130209.
Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.
嗜热栖热芽孢杆菌乳酸脱氢酶是已知最耐热的细菌酶之一，其三维结构已被解析，编码该酶的基因已被克隆，并且该蛋白质能够很容易地过量表达。已制备了该酶的两种突变体。在一种突变体中，精氨酸171被替换为色氨酸（R171W），谷氨酰胺102被替换为精氨酸（Q102R）。在另一种突变体中，保留了突变Q102R，但精氨酸171被替换为酪氨酸（R171Y）。此外，还存在一个意外产生的C97G突变体。两种突变体均通过室温下的悬滴气相扩散法结晶。在50 mM哌嗪盐酸盐缓冲液中，以硫酸铵为沉淀剂获得了双金字塔形晶体。这些晶体属于空间群P6(2)22（P6(4)22）（而天然酶，其结构已由Piontek等人解析，《蛋白质》7:74 - 92，1990）结晶于空间群P6(1)），对于R171W、Q102R、C97G三重突变体，a = 102.3 Å，c = 168.6 Å；对于R171Y、Q102R、C97G突变体，a = 98.2 Å，c = 162.1 Å。这些晶体形式在不对称单元中似乎包含四分之一的四聚体（相对分子质量135,000），VM值分别为3.8和3.3 ų/道尔顿。R171W突变体的衍射分辨率为2.5 Å，R171Y突变体的衍射分辨率约为3.5 Å。
