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Characterization of 2,3-dihydroxybiphenyl 1,2-dioxygenase, a key enzyme in polychlorinated biphenyls (PCBs) biodegradation

University of British Columbia

2,3-Dihydroxybiphenyl 1,2-dioxygenase (DHBD[sub LB400], EC 1.13.11.39) from Burkholderia sp. LB400, the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The steady-state kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsoryorder ternary-complex mechanism in which substrate inhibition occurs. DHBD[sub LB400] was also stabilized by small organic molecules. The molecules that stabilized DHBD[sub LB400] most effectively also inhibited the cleavage reaction most strongly. DHBD[sub LB400] was subject to reversible substrate inhibition and mechanism-based inactivation. Analysis of the mechanism-based inactivation revealed that it was similar to the O₂-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O₂ to promote ring-cleavage, but also increased the rate of 0₂-dependent inactivation. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme:catecholic:02 ternary complex. To evaluate the role of DHBDs in the degradation of polychlorinated biphenyls (PCBs), the ability of DHBD[sub LB400] and two evolutionarily divergent isozymes (DHBD[sub p6]-I and DHBD[sub p6]-III) from Rhodococcus globerulus P6 to cleave each of the six monochlorinated DHBs was studied. DHBD[sub LB400]cleaved these compounds with specificities between 0.06 and 0.3 times that of unchlorinated DHB. In contrast to DHBD[sub LB400]> both rhodococcal enzymes had higher apparent specificity constants for some chlorinated DHBs. Interestingly, DHBD[sub LB400]and DHBD[sub P6]-I had a very poor reactivity towards 2'-Cl DHB, and were more susceptible to mechanism-based inactivation in its presence. Subsequent work with 2',6'-diCl DHB revealed that this compound was cleaved extremely slowly relative to DHB. It was found that 2',6'-diCl DHB competitively inhibited the cleavage of DHB with a K[sup app][sub ic]= 7 ± 1 nM; 0.14 ± 0.01 μM and 2.6 ± 0.2 μM for DHBD[sub LB400], DHBD[sub P6] - I and DHBD[sub p6]-III respectively. These data were shown to be in good agreement with the crystal structures of the DHBD[sub LB400]:2'-Cl DHB and DHBD[sub LB400]:2',6'- diCl DHB complexes (Dai et al. 2002, In preparation) that show that the chlorinated DHBs fit the active site very well and that the oriho chloro substituents partially occlude the putative 0₂-binding pocket, thereby inhibiting 0₂-binding as well as the reaction between the activated oxygen and catecholic species in the enzyme ternary complex. Finally, UV/vis spectroscopy was used to probe the nature of the binding of DHB and 3-nitrocatechol to DHBD[sub LB400]- The electronic absorption data demonstrate that DHBD[sub LB400] binds its catecholic substrate as a monoanion, confirming this feature of the proposed mechanism of extradiol dioxygenases. This conclusion is supported by UV resonance Raman spectroscopic data and a crystal structure of the DHBD[sub LB400]:DHB complex at 2.0 Å resolution (Vaillancourt et al. 2002, J. Am. Chem. Soc. 124, 2485-2496), which suggests that the substrate's 2-hydroxyl substituent, and not the 3-hydroxyl group, deprotonates upon binding.

Thesis/Dissertation

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2009-10-05

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http://hdl.handle.net/2429/13567

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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