title: Directed evolution of a ring-cleaving dioxygenase for polychlorinated biphenyl degradation.
abstract: DoxG, an extradiol dioxygenase involved in the aerobic catabolism of naphthalene, possesses a weak ability to cleave 3,4-dihydroxybiphenyls (3,4-DHB), critical polychlorinated biphenyl metabolites. A directed evolution strategy combining error-prone PCR, saturation mutagenesis, and DNA shuffling was used to improve the polychlorinated biphenyl-degrading potential of DoxG. Screening was facilitated through analysis of filtered, digital imaging of plated colonies. A simple scheme, which is readily adaptable to other activities, enabled the screening of >10(5) colonies/h. The best variant, designated DoxGSMA2, cleaved 3,4-DHB with an apparent specificity constant of 2.0 +/- 0.3 x 10(6) m(-1) s(-1), which is 770 times that of wild-type (WT) DoxG. The specificities of DoxGSMA2 for 1,2-DHN and 2,3-DHB were increased by 6.7-fold and reduced by 2-fold, respectively, compared with the WT enzyme. DoxGSMA2 contained three substituted residues with respect to the WT enzyme: L190M, S191W, and L242S. Structural data indicate that the side chains of residues 190 and 242 occur on opposite walls of the substrate binding pocket and may interact directly with the distal ring of 3,4-DHB or influence contacts between this substrate and other residues. Thus, the introduction of two bulkier residues on one side of the substrate binding pocket and a smaller residue on the other may reshape the binding pocket and alter the catalytically relevant interactions of 3,4-DHB with the enzyme and dioxygen. Kinetic analyses reveal that the substitutions are anti-cooperative.
authors.affiliation: Department of Microbiology and Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Pascal D
authors.initials: PD
authors.lastname: Fortin
authors.forename: Iain
authors.initials: I
authors.lastname: MacPherson
authors.forename: David B
authors.initials: DB
authors.lastname: Neau
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Bolin
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D049308:Dioxygenases / Q000378:metabolism*; D019020:Directed Molecular Evolution; D007700:Kinetics; D008958:Models, Molecular; D011078:Polychlorinated Biphenyls / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011078:Polychlorinated Biphenyls; D049308:Dioxygenases