Rational design of catechol-2, 3-dioxygenase for improving the enzyme characteristics.
Catechol-2, 3-dioxygenase (C23O) from Pseudomonas sp. CGMCC2953 identified in our laboratory, which is one of the key enzymes responsible for phenanthrene biodegradation, was expected to get better characteristics tolerant to environment for its further application. With the aim of improving the enzyme properties by introducing intermolecular disulfide bonds, X-ray structure of a C23O from Pseudomonas putida MT-2, a highly conserved homologous with the C23O from Pseudomonas sp. CGMCC2953, was directly used to find the potential sites for forming disulfide bonds between two monomers of the target C23O. Two sites, Ala229 and His294, were identified and mutated to cysteine, respectively, by using site mutagenesis. The expected disulfide bond between these two CYS residues was confirmed with both molecular modeling and experimental results. The optimum temperature of the mutated enzyme was widened from 40 to 40 approximately 50 degrees C. The mutated C23O became more alkalescency stable compared with the wild-type enzyme, e.g., 75% of the maximal enzyme activity retained even under pH 9.5 while 50% residue for the wild-type one. Improvement of thermostability of the mutated C230 with the redesigned disulfide was also confirmed.
Wei
Jiashi
J
School of Life Science, Fudan University, Shanghai 200433, People's Republic of China. hugh.w@163.com
Zhou
Ying
Y
Xu
Tao
T
Lu
Baorong
B
D000595:Amino Acid Sequence; D050557:Catechol 2,3-Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D020407:Internet; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology; D013696:Temperature
D004220:Disulfides; D050557:Catechol 2,3-Dioxygenase