Expression, Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the Mutant Pro(229)Ser of Thermostable Catechol 2,3-dioxygenase.
The mutant Pro(229)Ser of thermostable catechol 2,3-dioxygenase (TC23O) was expressed and purified. Enzymatic analysis revealed that its thermostability was decreased, the temperature corresponding to 50% enzyme activity being about 10.2 degrees lower than that of the wild type TC23O. Its kinetic parameter k(cat)/K(m) value (4.89x 10(6) mol(-1).s(-1)) was lower than that of the wild type TC23O(6.97x10(6) mol(-1).s(-1)). By the hanging-drop vapor-diffusion method using polyethylene glycol 400 as a precipitant, the mutant Pro(229)Ser of TC23O crystallizedat 4 degrees. X-ray diffraction analysis revealed that the crystals belong to the orthorhombic space group I(222) with unit-cell parameters a 7.059 nm, b 7.415 nm, c 13.311 nm, and they diffracted to at least 0.24 nm resolution. Assuming the presence of 2 molecules of the mutant Pro(229)Ser in the asymmetric unit, the Matthews parameter (Vm) was calculated to be 2.49x10( 3) nm(3).D(-1), and the solventcontent was about 51%. The crystal structure determination is now in progress.
Jiang
Tao
T
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, China. ymmao@fudan.edu.cn
Ji
Chao-Neng
CN
Sheng
Xiao-Yu
XY
Mao
Yu-Min
YM