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J. Biol. Chem., Vol. 280, Issue 51, 42307-42314, December 23, 2005

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Directed Evolution of a Ring-cleaving Dioxygenase for Polychlorinated Biphenyl Degradation^*

Pascal D. Fortin1, Iain MacPherson2, David B. Neau3, Jeffrey T. Bolin, and Lindsay D. Eltis4

From the Departments of Microbiology and Biochemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2054

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 >10⁵ colonies/h. The best variant, designated DoxG_SMA2, cleaved 3,4-DHB with an apparent specificity constant of 2.0 ± 0.3 x 10⁶ M^-1 s^-1, which is 770 times that of wild-type (WT) DoxG. The specificities of DoxG_SMA2 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. DoxG_SMA2 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.

Received for publication, September 23, 2005 , and in revised form, October 11, 2005.

^* This work was supported in part by Operating and Strategic grants from the Natural Sciences and Engineering Research Council of Canada and by National Institutes of Health Grant R01-GM52381. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of Fonds pour la Formation de Chercheurs et l'Aide à la Recherche and Natural Sciences and Engineering Research Council of Canada postgraduate scholarships.

² Recipient of Natural Sciences and Engineering Research Council of Canada postgraduate scholarship.

³ Supported in part by an National Institutes of Health institutional training award, T32-GM008296. Current address: Center for Advanced Microstructures and Devices, Louisiana State University, 6980 Jefferson Hwy., Baton Rouge, LA 70806.

⁴ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of British Columbia, 1365-2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-0042; Fax: 604-822-6041; E-mail: leltis{at}interchange.ubc.ca.

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