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Charcterization and preliminary X-ray diffraction analysis of the mutant Pro229Ser of thermostable catechol 2,3-dioxygenase

[ Vol. 8 , Issue. 4 ]

Author(s):

Tao Jiang, Chao-Neng Ji, Xiao-Yu Sheng, yi Xie and Yu-Min Mao   Pages 327 - 332 ( 6 )

Abstract:


Catechol 2,3-dioxygenase (C23O) is one of extradiol type dioxygenase that cleave the aromatic C-C bond at meta position of the hazardous dihydroxylated aromatic substrates, and plays a key role in the degradation of aromatic molecules in the environment by soil bacteria [1,2]. In a high temperature environment, the counterpart of the mesophilic C23O is the thermostable catechol 2,3-dioxygenase (TC23O) from thermophiles. The pheB gene encoding for TC23O from the thermophile Bacillus stearothermophilus FDTP-3 has been cloned [3], and subsequently has been subcloned into the plasmid pJLA503, and over expressed in E. coli TG 1 by our lab. Analytical results reveal that TC23O is a homotetramer with an apparent molecular mass of 140 kD, and each subunit is composed of 327 amino acid residues. The homology in amino acid sequence between the mesophilic C23O [2] and the thermophilic TC23O is about 27 percent. TC23O is an ideal model protein for exploring the mechanism and structural basis for protein thermos tability owing to the ease and high sensitivity in its assay. In order to investigate the effects of amino acid replacement on the thermostability of TC23O, random PCR mutagenesis was utilized to generate amino acid substituted mutants with changed thermostability. Pro229Ser is one of these mutants, the thermostability of which is decreased 10.2 °C compared with that of the wild type TC23O, which implies that Pro229 is probably important for the thermostability of TC23O. As a preliminary step in the study of the molecular basis of thermostability, we report here the expression, purification, characterization, crystallization and preliminary X-ray diffraction analysis of Pro229Ser.

Keywords:

mutant Pro229Ser, catechol 2,3-dioxygenase, Bacillus stearothermophilus

Affiliation:

State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China



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