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Please use this identifier to cite or link to this item: http://repository.iitr.ac.in/handle/123456789/26955
Title: Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution
Authors: Kumar, Pravindra R.Manish
Mohammadi M.
Viger J.-F.
Barriault D.
Gomez-Gil L.
Eltis L.D.
Bolin J.T.
Sylvestre M.
Published in: Journal of Molecular Biology
Abstract: The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE LB400) are largely unknown. BphAEp4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAELB400, yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAELB400 and BphAEp4 and examine the biochemical properties of two BphAELB400 variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity. © 2010 Elsevier Ltd. All rights reserved.
Citation: Journal of Molecular Biology, 405(2): 531-547
URI: https://doi.org/10.1016/j.jmb.2010.11.009
http://repository.iitr.ac.in/handle/123456789/26955
Issue Date: 2011
Publisher: Academic Press
Keywords: Burkholderia xenovorans LB400
enzyme engineering
PCB
polychlorinated biphenyl
Rieske-type oxygenase
ISSN: 222836
Author Scopus IDs: 55064809000
23390544500
37038864200
6602879821
18233632900
7003863696
57197844041
7005984229
Author Affiliations: Kumar, P., Department of Biological Sciences, Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States, Department of Biotechnology, Indian Institute of Technology, Roorkee-247667, India
Mohammadi, M., Institut National de la Recherche Scientifique (INRS-Institut Armand-Frappier), Laval, QC H7V 1B7, Canada
Viger, J.-F., Institut National de la Recherche Scientifique (INRS-Institut Armand-Frappier), Laval, QC H7V 1B7, Canada
Barriault, D., Institut National de la Recherche Scientifique (INRS-Institut Armand-Frappier), Laval, QC H7V 1B7, Canada
Gomez-Gil, L., Departments of Microbiology and Biochemistry, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
Eltis, L.D., Departments of Microbiology and Biochemistry, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
Bolin, J.T., Department of Biological Sciences, Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States
Sylvestre, M., Institut National de la Recherche Scientifique (INRS-Institut Armand-Frappier), Laval, QC H7V 1B7, Canada
Funding Details: This work was supported by Discovery and Strategic grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) . Use of the BioCARS Sector 14 at the Advanced Photon Source (APS), Argonne National Laboratory, was supported by the National Institutes of Health, National Center for Research Resources, under grant number RR007707. Additional X-ray diffraction data were collected at APS using Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline; supporting institutions may be found at http:// www.ser-cat.org/members.html . Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. National Institutes of Health, NIH; U.S. Department of Energy, USDOE; National Center for Research Resources, NCRR: P41RR007707; Office of Science, SC; Argonne National Laboratory, ANL; Natural Sciences and Engineering Research Council of Canada, NSERC
Corresponding Author: Sylvestre, M.; Institut National de la Recherche Scientifique (INRS-Institut Armand-Frappier), Laval, QC H7V 1B7, Canada; email: Michel.Sylvestre@iaf.inrs.ca
Appears in Collections:Journal Publications [BT]

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