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J. Bacteriol. doi:10.1128/JB.01476-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Characterization of Biphenyl Dioxygenase of Pandoraea pnomenusa B-356 as a Potent Polychlorinated Biphenyl-Degrading Enzyme

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

^* To whom correspondence should be addressed. Email: leltis{at}interchange.ubc.ca.

	Abstract

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO_B356 from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO_LB400 from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming each of 9 congeners in the following order of preference: 2,3',4-triCl 2,3,4'-triCl > 3,3'-diCl > 2,4,4'-triCl > 4,4'-diCl 2,2'-diCl > 2,6-diCl > 2,2',3,3'-tetraCl 2,2',5,5'-tetraCl. Except for 2,2',5,5'-tetraCl biphenyl, BPDO_B356 transformed each at higher rates than BPDO_LB400. The assays used either whole cells or purified enzymes, and either individual congeners or mixtures thereof. Product analyses established previously unrecognized BPDO_B356 activities, including the 3,4-dihydroxylations of 2,6-diCl biphenyl. BPDO_LB400 had a greater apparent specificity for biphenyl than BPDO_B356 (k_cat/K_m = 2.4 (0.7) x 10⁶ M^-1s^-1 vs. 0.21 (0.04) x 10⁶ M^-1s^-1). However, the latter transformed biphenyl at a higher maximal rate (k_cat = 4.1 (0.2) vs. 0.4 (0.1) s^-1). A variant of BPDO_LB400 containing 4 active-site residues of BPDO_B356 transformed para-substituted congeners better than BPDO_LB400. 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 the 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. The crystal structure of the BPDO_B356:2,6-diCl biphenyl complex at 2.4 Å resolution, the first 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 respective BPDOs.

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