| Summary: | 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. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
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