title: Altering substrate specificity of catechol 2,3-dioxygenase from Planococcus sp. strain S5 by random mutagenesis.
abstract: c23o gene, encoding catechol 2,3-dioxygenase from Planococcus sp. strain S5 was randomly mutagenized to generate variant forms of the enzyme with higher degradation activity. Additionally, the effect of introduced mutations on the enzyme structure was analyzed based on the putative 3D models the wild-type and mutant enzymes. C23OB58 and C23OB81 mutant proteins with amino acid substitutions in close proximity to the enzyme surface or at the interface and in the vicinity of the enzyme active site respectively showed the lowest activity towards all catecholic substrates. The relative activity of C23OC61 mutant towards para-substituted catechols was 20-30% lower of the wild-type enzyme. In this mutant all changes: F191I, C268R, Y272H, V280A and Y293D were located within the conserved regions of C-terminal domain. From these F191I seems to have significant implications for enzyme activity. The highest activity towards different catechols was found for mutant C23OB65. R296Q mutation improved the activity of C23O especially against 4-chlorocatechol. The relative activity of above-mentioned mutant detected against this substrate was almost 6-fold higher than the wild-type enzyme. These results should facilitate future engineering of the enzyme for bioremediation.
authors.affiliation: Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Katowice, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Hupert-Kocurek
authors.affiliation: Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Katowice, Poland.
authors.forename: Danuta
authors.initials: D
authors.lastname: Wojcieszyńska
authors.affiliation: Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Katowice, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Guzik
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D050557:Catechol 2,3-Dioxygenase / Q000737:chemistry / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism; D016296:Mutagenesis; D058642:Planococcus Bacteria / Q000201:enzymology*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002396:Catechols; D050557:Catechol 2,3-Dioxygenase; C034221:catechol