Structural Insight into the Expanded PCB-Degrading Abilities of a Biphenyl Dioxygenase Obtained by Directed Evolution

JOURNAL OF MOLECULAR BIOLOGY

卷:405

Article

Kumar, Pravindra^2,3,6  Mohammadi, Mahmood¹  Viger, Jean-Francois¹  Barriault, Diane¹  Gomez-Gil, Leticia^4,5  Eltis, Lindsay D.^4,5  Bolin, Jeffrey T.^2,3  Sylvestre, Michel¹ 

[1] Inst Natl Rech Sci INRS Inst Armand Frappier, Laval, PQ H7V 1B7, Canada

[2] Purdue Univ, Dept Biol Sci, W Lafayette, IN 47907 USA

[3] Purdue Univ, Ctr Canc Res, W Lafayette, IN 47907 USA

[4] Univ British Columbia, Inst Life Sci, Dept Microbiol, Vancouver, BC V6T 1Z3, Canada

[5] Univ British Columbia, Inst Life Sci, Dept Biochem, Vancouver, BC V6T 1Z3, Canada

[6] Indian Inst Technol, Dept Biotechnol, Roorkee 247667, Uttar Pradesh, India

关键词: polychlorinated biphenyl;    Burkholderia xenovorans LB400;    enzyme engineering;    Rieske-type oxygenase;    PCB;   

DOI  :  10.1016/j.jmb.2010.11.009

来源: Elsevier

【 摘 要 】

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. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) 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. (C) 2010 Elsevier Ltd. All rights reserved.

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