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Identification of molecular determinants of substrate specificity and activity for biphenyl dioxygenase from Comamonas testosteroni B-356

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Identification of molecular determinants of substrate specificity and activity for biphenyl dioxygenase from Comamonas testosteroni B-356

Agar, Nathalie Yvonne Rachel (2002) Identification of molecular determinants of substrate specificity and activity for biphenyl dioxygenase from Comamonas testosteroni B-356. PhD thesis, Concordia University.

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Abstract

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Identification of molecular determinants for BPDO substrate selectivity and activity should allow engineering it to accept a range of chlorinated biphenyls for bioremediation. Highly active BPDO has been purified, and crystallized under anaerobic conditions. The first crystal structures of BPDO, and its substrate-bound and product-bound binary complexes, have been solved. Steady-state kinetics assays monitoring O 2 consumption demonstrated that BPDO transformed biphenyl and dichlorobiphenyls with the following order of apparent specificities: biphenyl > 3,3 ' - > 2,2 ' - > 4,4 ' -dichlorobiphenyl, but with specificity constants all within a factor of 3. The ability of the enzyme to utilize O 2 depended strongly on the biphenyl substrate, with specificity constants for oxygen over one order of magnitude smaller in the presence of the dichlorinated biphenyls compared to biphenyl. Moreover, the selected dichlorobiphenyls were found to be partial uncouplers of the reaction, reducing O 2 to H 2 O 2 . Based on the high-resolution crystal structure of BPDO and its binary complexes, the roles of some specific residues were investigated by site-directed mutagenesis. Moreover, three BPDOs sharing between 70-95% sequence identity were overexpressed, purified in highly active forms, and compared in terms of steady-state kinetic parameters for biphenyls, as well as uncoupling behaviour. BPDO comprises two metal centers required for enzymatic activity: a catalytic mononuclear iron and a [2Fe-2S] Rieske-type cluster for electron transfer. To overexpress holo-BPDOs, the iron-sulfur cluster ( isc ) assembly genes from Pseudomonas aeruginosa PA01 were cloned and used for coexpression. The Fe-S cluster of BPDO was shown to be sensitive to reactive oxygen species such as superoxide, but the isc gene products appeared to have the ability to repair damaged clusters in crude extract reconstitution assays. The experimental approaches and results described in this thesis contribute to the identification of structural features important for substrate selectivity, and ultimately may be useful for construction of modified BPDOs with enhanced activity on chlorinated biphenyls

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Agar, Nathalie Yvonne Rachel
Pagination:xx, 237 leaves : col. ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (Ph.D.)
Program:Chemistry and Biochemistry
Date:2002
Thesis Supervisor(s):Powlowski, Justin B
ID Code:1841
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:22
Last Modified:08 Dec 2010 10:23
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