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

2,3-Dihydroxybiphenyl 1,2-dioxygenase (DHBD_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 compulsory-order ternary-complex mechanism in which substrate inhibition occurs. DHBD_LB400 was also stabilized by small organic molecules. The molecules that stabilized DHBD _LB400 most effectively also inhibited the cleavage reaction most strongly. DHBD_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). The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme:catecholic:O₂ ternary complex.; To evaluate the role of DHBDs in the degradation of polychlorinated biphenyls (PCBs), the ability of DHBD_LB400 and two evolutionarily divergent isozymes (DHBD_P6-I and DHBD_P6-III) from Rhodococcus globerulus P6 to cleave each of the six monochlorinated DHBs was studied. DHBD_LB400 cleaved these compounds with specificities between 0.06 and 0.3 times that of unchlorinated DHB. In contrast to DHBD_LB400 , both rhodococcal enzymes had higher apparent specificity constants for some chlorinated DHBs. Interestingly, DHBD_LB400 and DHBD _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 $Kappic$ = 7 ± 1 nM; 0.14 ± 0.01 μM and 2.6 ± 0.2 μM for DHBD_LB400, DHBD_P6-I and DHBD_P6-III respectively. These data were shown to be in good agreement with the crystal structures of the DHBD_LB400:2′-Cl DHB and DHBD_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 ortho chloro substituents partially occlude the putative O₂-binding pocket, thereby inhibiting O₂-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_LB400. (Abstract shortened by UMI.)...