Expression,
Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the
Mutant Pro229Ser of Thermostable Catechol 2,3-dioxygenase
JIANG Tao, JI Chao-Neng, SHENG Xiao-Yu, MAO Yu-Min*
( State Key Laboratory of Genetic Engineering, Institute of Genetics,
School of Life Science, Fudan University, Shanghai 200433, China
)
Abstract The mutant
Pro229Ser 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 ℃ lower than that of the wild type TC23O. Its kinetic parameter kcat/Km
value (4.89 ×106 mol－1・s－1) was lower than that of the
wild type TC23O(6.97×106 mol－1・s－1). By the hanging-drop
vapor-diffusion method using polyethylene glycol 400 as a precipitant, the
mutant Pro229Ser of TC23O crystallizedat 4 ℃. X-ray diffraction
analysis revealed that the crystals belong to the orthorhombic space group I222
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 Pro229Ser in the asymmetric unit, the Matthews parameter (Vm)
was calculated to be 2.49×10－3 nm3・D－1, and the solventcontent was
about 51%. The crystal structure determination is now in progress.
Key words thermostable enzyme； X-ray diffraction analysis； thermostability； thermostable catechol
2,3-dioxygenase
*Corresponding author： Tel, 86-21-65643958； Fax, 86-21-65642502； e-mail, [email protected]