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J. Biol. Chem., Vol. 281, Issue 41, 30933-30940, October 13, 2006

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Structures of Ternary Complexes of BphK, a Bacterial Glutathione S-Transferase That Reductively Dechlorinates Polychlorinated Biphenyl Metabolites^*

From the Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Prokaryotic glutathione S-transferases are as diverse as their eukaryotic counterparts but are much less well characterized. BphK from Burkholderia xenovorans LB400 consumes two GSH molecules to reductively dehalogenate chlorinated 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), inhibitory polychlorinated biphenyl metabolites. Crystallographic structures of two ternary complexes of BphK were solved to a resolution of 2.1Å. In the BphK-GSH-HOPDA complex, GSH and HOPDA molecules occupy the G- and H-subsites, respectively. The thiol nucleophile of the GSH molecule is positioned for S_N2 attack at carbon 3 of the bound HOPDA. The respective sulfur atoms of conserved Cys-10 and the bound GSH are within 3.0Å_, consistent with product release and the formation of a mixed disulfide intermediate. In the BphK-(GSH)₂ complex, a GSH molecule occupies each of the two subsites. The three sulfur atoms of the two GSH molecules and Cys-10 are aligned suitably for a disulfide exchange reaction that would regenerate the resting enzyme and yield disulfide-linked GSH molecules. A second conserved residue, His-106, is adjacent to the thiols of Cys-10 and the GSH bound to the G-subsite and thus may stabilize a transition state in the disulfide exchange reaction. Overall, the structures support and elaborate a proposed dehalogenation mechanism for BphK and provide insight into the plasticity of the H-subsite.

Received for publication, April 3, 2006 , and in revised form, August 11, 2006.

The atomic coordinates and structure factors (code 2DSA and 2GDR) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Discovery Grants from the Natural Sciences and Engineering Research Council (to L. D. E. and M. E. P. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures.

¹ Recipient of a University Graduate Fellowship from the University of British Columbia.

² Recipient of Fonds pour la Formation de Chercheurs et l'Aide à la Recherche and Natural Sciences and Engineering Research Council postgraduate scholarships.

³ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver BC Canada V6T 1Z3. Tel.: 604-822-8022; Fax: 604-822-6041; E-mail: michael.murphy{at}ubc.ca.

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