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Degradation of polychlorinated biphenyl (PCB) metabolites : directed evolution and enzymatic fortuity

University of British Columbia

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 enzyme catalyzes the glutathione-dependant dehalogenation of certain inhibitory CI HOPDAs. BphK catalyzed the dechlorination of 3-Cl HOPDA with a specificity constant of ~10⁴ M⁻¹s⁻¹ in a reaction that utilized a ternary complex mechanism and 2 equivalents of GSH. The identified product of the reaction, HOPDA, is the substrate of the hydrolase from the bph pathway. The relatively low specificity constant of BphK for 3-Cl HOPDAs corroborate genetic evidence that the enzyme was recently recruited to the bph pathway to facilitate PCB degradation.

Text

2010-01-06

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http://hdl.handle.net/2429/17659

Microbiology and Immunology

University of British Columbia

UBCV

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