[Effects of Pro229-->Ser and Glu243-->Gly on the characters of thermostable catechol 2, 3-dioxygenase].
In order to investigate the effects of amino acid replacement on the characters of thermostable catechol 2, 3-dioxygenase, two mutants (Pro229Ser and Glu243Gly) of this enzyme were obtained by using the method of PCR random mutagenesis. The wild type thermostable catechol 2, 3-dioxygenase and these two mutants (Pro229Ser, Glu243Gly) were over expressed in E. coli TG1 and purified. The enzymatic characters and thermostability of the wild type enzyme and the two mutants (Pro229Ser, Glu243Gly) were analyzed. The results revealed that the optimum enzymatic temperature of the two mutants were the same as that of the wild type enzyme (60 degrees C) and the Kcat/Km value of Pro229Ser and Glu243Gly (4.89 +/- 0.01 x 10(6) mol-1 s-1 and 5.88 +/- 0.01 x 10(6) mol-1 s-1, respectively) were reduced compared with the wild type enzyme (6.97 +/- 0.01 x 10(6) mol-1 s-1). However, the thermostability of Pro229Ser extremely decreased 10.2 degrees C and the thermostability of Glu243Gly slightly increased 1.5 degrees C. It was proposed that Pro229 played an important role on the thermostability of thermostable catechol 2, 3-dioxygenase.
State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China.
T
T
Jiang
C N
CN
Ji
X Y
XY
Sheng
Y M
YM
Mao
D000595:Amino Acid Sequence; D002384:Catalysis; D050557:Catechol 2,3-Dioxygenase; D049308:Dioxygenases; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D013329:Structure-Activity Relationship
D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase