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Journal of Bacteriology, August 2007, p. 5705-5715, Vol. 189, No. 15
0021-9193/07/$08.00+0     doi:10.1128/JB.01476-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Biphenyl Dioxygenase of Pandoraea pnomenusa B-356 As a Potent Polychlorinated Biphenyl-Degrading Enzyme{triangledown}

Leticia Gómez-Gil,1 Pravindra Kumar,2 Diane Barriault,3 Jeffrey T. Bolin,2 Michel Sylvestre,3 and Lindsay D. Eltis1*

Departments of Microbiology and Biochemistry, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada,1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana,2 Institut National de Recherche Scientifique (INRS-Institut Armand-Frappier), Pointe-Claire, Quebec H9R 1G6, Canada3

Received 18 September 2006/ Accepted 11 May 2007

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDOB356 from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDOLB400 from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro ~ 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro ~ 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro ~ 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDOB356 transformed each congener at a higher rate than BPDOLB400. The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDOB356 activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDOLB400 had a greater apparent specificity for biphenyl than BPDOB356 (kcat/Km = 2.4 x 106 ± 0.7 x 106 M–1 s–1 versus kcat/Km = 0.21 x 106 ± 0.04 x 106 M–1 s–1). However, the latter transformed biphenyl at a higher maximal rate (kcat = 4.1 ± 0.2 s–1 versus kcat = 0.4 ± 0.1 s–1). A variant of BPDOLB400 containing four active site residues of BPDOB356 transformed para-substituted congeners better than BPDOLB400. Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O2 consumption was approximately proportional to congener depletion. At 2.4-Å resolution, the crystal structure of the BPDOB356-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.

* Corresponding author. Mailing address: University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Phone: (604) 822-0042. Fax: (604) 822-6041. E-mail: leltis{at}interchange.ubc.ca

{triangledown} Published ahead of print on 25 May 2007.

Journal of Bacteriology, August 2007, p. 5705-5715, Vol. 189, No. 15
0021-9193/07/$08.00+0     doi:10.1128/JB.01476-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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