The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation: LARGE 3-SUBSTITUENTS PREVENT TAUTOMERIZATION

doi:10.1074/jbc.M707035200

HOME

HELP

FEEDBACK

SUBSCRIPTIONS

The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation

LARGE 3-SUBSTITUENTS PREVENT TAUTOMERIZATION^*

Shiva Bhowmik¹, Geoff P. Horsman¹, Jeffrey T. Bolin², and Lindsay D. Eltis³

From the Purdue Cancer Center and Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2054 and the Departments of Biochemistry and Molecular Biology, and Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

The microbial degradation of polychlorinated biphenyls (PCBs)by the biphenyl catabolic (Bph) pathway is limited in part bythe pathway's fourth enzyme, BphD. BphD catalyzes an unusualcarbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoicacid (HOPDA), in which the substrate is subject to histidine-mediatedenol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAssuch as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentiallyhydrolyzed a series of 3-substituted HOPDAs in the order H >F > Cl > Me, suggesting that catalysis is affected bysteric, not electronic, determinants. Transient state kineticstudies performed using wild-type BphD and the hydrolysis-defectiveS112A variant indicated that large 3-substituents inhibitedHis-265-catalyzed tautomerization by 5 orders of magnitude.Structural analyses of S112A·3-Cl HOPDA and S112A·3,10-diFHOPDA complexes revealed a non-productive binding mode in whichthe plane defined by the carbon atoms of the dienoate moietyof HOPDA is nearly orthogonal to that of the proposed keto tautomerobserved in the S112A·HOPDA complex. Moreover, in the3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 Åfrom its position near the catalytic His-265 to hydrogen bondwith Arg-190 and access of His-265 is blocked by the 3-Cl substituent.Nonproductive binding may be stabilized by interactions involvingthe 3-substituent with non-polar side chains. Solvent moleculeshave poor access to C6 in the S112A·3-Cl HOPDA structure,more consistent with hydrolysis occurring via an acyl-enzymethan a gem-diol intermediate. These results provide insightinto engineering BphD for PCB degradation.

Received for publication, August 22, 2007 , and in revised form, October 3, 2007.

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

^* This work was supported by a grant from the Natural Sciencesand Engineering Research Council of Canada (Discovery grantto L. D. E.). X-ray diffraction data were collected at SE RegionalCollaborative Access Team (SER-CAT) 22-ID beamline at the AdvancedPhoton Source, Argonne National Laboratory. Use of the AdvancedPhoton Source was supported by the U. S. Dept. of Energy, Officeof Science, Office of Basic Energy Sciences, under ContractNo. W-31-109-Eng-38. The costs of publication of this articlewere defrayed in part by the payment of page charges. This articlemust therefore be hereby marked "advertisement" in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: 915 W. State St., West Lafayette, IN 47907. Tel.: 765-494-4408; Fax: 765-494-0876; E-mail: jtb{at}purdue.edu. ³ To whom correspondence may be addressed: 1365-2350, Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-0042; Fax: 604-822-6041; E-mail: leltis{at}interchange.ubc.ca.