Cloning and mutagenesis of catechol 2,3-dioxygenase gene from the gram-positive Planococcus sp. strain S5.
In this study, the catechol 2,3-dioxygenase gene that encodes a 307- amino-acid protein was cloned from Planococcus sp. S5. The protein was identified to be a member of the superfamily I, subfamily 2A of extradiol dioxygenases. In order to study residues and regions affecting the enzyme's catalytic parameters, the c23o gene was randomly mutated by error-prone PCR. The wild-type enzyme and mutants containing substitutions within either the C-terminal or both domains were functionally produced in Escherichia coli and their activity towards catechol was characterized. The C23OB65 mutant with R296Q substitution showed significant tolerance to acidic pH with an optimum at pH 5.0. In addition, it showed activity more than 1.5 as high as that of the wild type enzyme and its Km was 2.5 times lower. It also showed altered sensitivity to substrate inhibition. The results indicate that residue at position 296 plays a role in determining pH dependence of the enzyme and its activity. Lower activity toward catechol was shown for mutants C23OB58 and C23OB81. Despite lower activity, these mutants showed higher affinity to catechol and were more sensitive to substrate concentration than nonmutated enzyme.
Department of Biochemistry, Faculty of Biology and Environment Protection, University of Silesia in Katowice, Katowice, Poland.
Katarzyna
K
Hupert-Kocurek
Agnieszka
A
Stawicka
Danuta
D
Wojcieszyńska
Urszula
U
Guzik
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D050557:Catechol 2,3-Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058641:Planococcaceae / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D002396:Catechols; D004269:DNA, Bacterial; D050505:Mutant Proteins; D011994:Recombinant Proteins; D050557:Catechol 2,3-Dioxygenase; C034221:catechol