Degradation of polychlorinated biphenyl (PCB) metabolites : directed evolution and enzymatic fortuity
department or school: Science, Faculty of
department or school: Microbiology and Immunology, Department of
data provider: DSpace
degree campus: UBCV
creator: Fortin, Pascal
issued: 2010-01-06T22:38:31Z
issued: 2005
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: A multi-faceted approach was used to investigate and overcome the PCB-degrading limitations of the Bph enzymes, responsible for the catabolism of biphenyl. First, the reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia xenovorans sp. LB400 (DHBD[sub LB400]), DHBD[sub P6]-I and DHBD[sub P6]-III from Rhodococcus globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 (THBD[sub RW1]). The specificity of each enzyme for particular DHBs differed by up to 3 orders of magnitude. Moreover, each enzyme cleaved at least one of the tested chlorinated DHBs better than DHB. However, no enzyme was able to cleave 2',6'-diCl DHB or 3,4-DHB, two recalcitrant PCB metabolites. In the second facet of the study, biological selections were designed to facilitate the engineering of PCB-degrading enzymes via directed evolution. Although these selections failed to link the desired activities to host cell viability, they showed promise for other biocatalysts. In addition, a broadly useful high-throughput colorimetric screen was developed. The latter was applied to increase the specificity of DoxG, an extradiol dioxygenase from Pseudomonas sp. CI8, for 3,4-DHB. A single round of directed evolution yielded DOXG[sub SMA2], a variant that cleaved 3,4-DHB ∼1000-fold more specifically than the wild-type enzyme. DOXG[sub SMA2] contained three substituted residues: L190M, S191W and L242S. The crystal structure of the DoxG:3,4-DHB binary complex indicates that residues at position 190 and 242 occur on opposite sides of the DHB-binding pocket and may interact directly with the distal ring of the substrate. Kinetic analyses revealed that the substitutions are anti-cooperative. Finally, characterization of BphK, the glutathione S-transferase from the bph pathway of B. xenovorans sp. LB400, revealed that the e…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/17659
note: DEGRADATION OF POLYCHLORINATED BIPHENYL (PCB) METABOLITES: DIRECTED EVOLUTION AND E N Z Y M A T I C FORTUITY By Pascal Fortin B . S c , Universite Laval, 1999 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E STUDIES ( M I C R O B I O L O G Y A N D I M M U N O L O G Y ) The University of British Columbia September 2005 © Pascal Fortin, 2005 ABSTRACT A multi-faceted approach was used to investigate and overcome the PCB-degrading limitations of the Bph enzymes, responsible for the catabolism of biphenyl. First, the reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia xenovorans sp. LB400 (DHBDL B40o), D H B D P 6 - I and DHBD P 6 - I I I from Rhodococcus globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 ( T H B D R W i ) . The specificity of each enzyme for particular DHBs differed by up to 3 orders of magnitude. Moreover, each enzyme cleaved at least one of the tested chlorinated D H B s better than D H B . However, no enzyme was able to cleave 2' ,6 '-diCl D H B or 3,4-DHB, two recalcitrant PCB metabolites. In the second facet of the study, biological selections were designed to facilitate the engineering of PCB-degrading enzymes via directed evolution. Although these selections failed to link the desired activities to host cell viability, they showed promise for other biocatalysts. In addition, a broadly useful high-throughput colorimetric screen was developed. The latter was applied to increase the specificity of DoxG, an extradiol dioxygenase from Pseudomonas sp. CI8 , for 3,4-DHB. A single round of directed evolution yielded DOXGSMA2, a variant that cleaved 3,4-DHB -1000-fold more specifically than the wild-type enzy…
has type: Thesis/Dissertation
date issued: 2005-11
is shown at: 10.14288/1.0092530
language: eng
degree discipline: Microbiology and Immunology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/17659