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

Abstract

ABSTRACT 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.