Evolutionarily divergent extradiol dioxygenases possess higher specificities for polychlorinated biphenyl metabolites

Abstract

The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorinated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp. strain LB400 (DHBDLB400), DHBDP6-I and DHBDP6-III from Rhodococcus globerulus P6, and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. strain RW1 (THBDRW1). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the Kmapp values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorinated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3',5'-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophobic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHBDP6-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHBDP6-III than was DHB. Indeed, DHBDP6-III had the highest apparent specificity for 4,3',5'-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THBDRW1 had the highest specificity for 2'-Cl DHB and was at least five times more resistant to inactivation by 2'-Cl DHB, consistent with the similarity between the latter and 2,2',3-trihydroxybiphenyl. Nonetheless, THBDRW1 had the lowest specificity for 2',6'-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (kcatapp < 0.001 s(-1)).

FIG. 1.

Reaction catalyzed by DHBD and related enzymes. In the aerobic catabolism of biphenyl, DHBD catalyzes a reaction in which R = H. In the aerobic catabolism of dibenzofuran, THBD_RW1 catalyzes a reaction in which R = OH.

FIG. 2.

Steady-state cleavage of DHB by THBD_RW1. (A) Dependence of initial velocity on the concentration of DHB in air-saturated buffer. The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are K_m^app = 2.8 ± 0.4 μM and V = 64 ± 2 μM/min. (B) Dependence of initial velocity on the concentration of O₂ with 100 μM DHB. The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are K_mO2^app = 830 ± 100 μM and V = 200 ± 20 μM/min. All experiments were performed using air-saturated 100 mM phosphate buffer, pH 7.0, at 25°C. Initial velocities obtained on different days were normalized according to the amount of enzyme used in the assay.

FIG. 3.

Stereo view of the modeled active site of THBD_RW1 bound to 2′,6′-diCl DHB. The residues lining the substrate pocket and the bound DHB are represented in gray and black, respectively. The ligands of the ferrous iron (shown as a sphere) are His147, His209, and Glu260. The conserved catalytic residues are His191, His241, and Tyr250.