Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBD(LB400); EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2′,6′-dichloro-2,3-dihydroxybiphenyl (2′,6′-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBD(P6)-I and DHBD(P6)-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBD(LB400), both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBD(P6)-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3′-Cl DHB ∼ DHB ∼ 4′-Cl DHB > 2′-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBD(P6)-I were among the best for DHBD(P6)-III (4-Cl DHB > 5-Cl DHB ∼ 6-Cl DHB ∼ 3′-Cl DHB > DHB > 2′-Cl DHB ∼ 4′-Cl DHB; DHBD(P6)-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBD(P6)-I and 2′-Cl DHB for DHBD(P6)-III. As observed in DHBD(LB400), chloro substituents influenced the reactivity of the dioxygenases with O(2). For example, the apparent specificities of DHBD(P6)-I and DHBD(P6)-III for O(2) in the presence of 2′-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBD(P6)-I and DHBD(P6)-III shared the relative inability of DHBD(LB400) to cleave 2′,6′-diCl DHB (apparent catalytic constants of 0.088 ± 0.004 and 0.069 ± 0.002 s(−1), respectively). However, these isozymes had remarkably different apparent K(m) values for this compound (0.007 ± 0.001, 0.14 ± 0.01, and 3.9 ± 0.4 μM for DHBD(LB400), DHBD(P6)-I, and DHBD(P6)-III, respectively). The markedly different reactivities of DHBD(P6)-I and DHBD(P6)-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.