Characterization of extradiol dioxygenases from a polychlorinated biphenyl-degrading strain that possess higher specificities for chlorinated metabolites

Abstract

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBDLB400; 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 DHBDP6-I and DHBDP6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBDLB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBDP6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3'-Cl DHB approximately DHB approximately 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 DHBDP6-I were among the best for DHBDP6-III (4-Cl DHB > 5-Cl DHB approximately 6-Cl DHB approximately 3'-Cl DHB > DHB > 2'-Cl DHB approximately 4'-Cl DHB; DHBDP6-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 DHBDP6-I and 2'-Cl DHB for DHBDP6-III. As observed in DHBDLB400, chloro substituents influenced the reactivity of the dioxygenases with O2. For example, the apparent specificities of DHBDP6-I and DHBDP6-III for O2 in the presence of 2'-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBDP6-I and DHBDP6-III shared the relative inability of DHBDLB400 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 micro M for DHBDLB400, DHBDP6-I, and DHBDP6-III, respectively). The markedly different reactivities of DHBDP6-I and DHBDP6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.

FIG. 1.

Reaction catalyzed by DHBD.

FIG. 2.

General mechanism of DHBD during steady-state turnover. Only DHBD_P6-I is subject to substrate inhibition. E* denotes inactivated forms of the enzyme. The rate constants j₁ and j₃ are associated with reactions that result in enzyme inactivation.

FIG. 3.

Steady-state cleavage of 6-Cl DHB by DHBD_P6-I and 4-Cl DHB by DHBD_P6-III. The experiments were performed using air-saturated potassium phosphate buffer, pH 7.0 (I = 0.1), at 25°C. (A) DHBD_P6-I-catalyzed cleavage of 6-Cl DHB. The line represents a best fit of the substrate inhibition equation to the data. The fitted parameters are KmAapp = 1.9 ± 0.3 μM, KiAapp = 3.0 ± 0.9 mM, and Vmaxapp = 41.1 ± 1.2 μM/min. The inset shows the initial portion of the graph (0 to 30 μM 6-Cl DHB) in more detail. (B) DHBD_P6-III-catalyzed cleavage of 4-Cl DHB. The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are KmAapp = 1.8 ± 0.2 μM and v = 52.7 ± 1.2 μM/min. The inset shows the initial portion of the graph (0 to 30 μM 4-Cl DHB) in more detail.

FIG. 4.

Steady-state utilization of 2′,6′-diCl DHB by DHBD_P6-I and DHBD_P6-III. The experiments were performed using air-saturated potassium phosphate buffer, pH 7.0 (I = 0.1), at 25°C. (A) DHBD_P6-I-catalyzed cleavage of DHB in the presence of 2′,6′-diCl DHB. The rate of DHB cleavage was determined by using 3.1 μM (□), 5.4 μM (▪), 9.3 μM (▵), 13.1 μM (○), 16.9 μM (+), 54 μM (•), and 82.9 μM (×) DHB. Fits were obtained using an equation similar in form to that describing competitive inhibition as described in Materials and Methods. The fit of the equation to the data yielded the following parameters: KmAapp = 0.144 ± 0.014 μM, KmDHBapp = 3.4 ± 0.3 μM, and Vmaxapp = 60.4 ± 1.5 μM/min. (B) The DHBD_P6-III-catalyzed cleavage of 2′,6′-diCl DHB. The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are Kmaxapp = 3.9 ± 0.4 μM and Vmaxapp = 3.0 ± 0.1 μM/min. The inset shows the initial portion of the graph (0 to 50 μM 2′,6′-diCl DHB) in more detail.