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Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO KF1 from Comamonas testosteroni KF1 and found that it had an apparent k cat / K m for phthalate of 0.58 ± 0.09 μM −1 s −1 , over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α 3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO KF1 larger than that of other characterized ROs. Complexes of PDO KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

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Biphenyl dioxygenase (BPDO), which is a Rieske-type oxygenase (RO) catalyzes the initial dioxygenation of biphenyl and some polychlorinated biphenyls (PCBs). In order to enhance the degradation ability of BPDO in terms of broader substrate range, BphAES283M, BphAEp4-S283M and BphAERR41-S283M variants were created from parent enzymes BphAELB400, BphAEp4 and BphAERR41, respectively by substitution at one residue, Ser283Met. The results of steady-state kinetic parameters show that for biphenyl, the kcat/Km value of BphAES283M, BphAEp4-S283M and BphAERR41-S283M significantly increased compared to their parent enzyme. Meanwhile, we determined the Steady-state kinetics of BphAEs toward highly chlorinated biphenyls. The results suggested the Ser283Met substitution enhanced the catalytic activity of BphAEs toward 2,3′,4,4′-CB, 2,2′,6,6′-CB and 2,3′,4,4′,5-CB. We compared the catalytic reaction of BphAELB400 and its variants toward 2,2′-CB, 2,5-CB and 2,6-CB. The biochemical data indicate that the Ser283Met substitution alters the orientation of the substrate inside the catalytic site, thereby its site of hydroxylation. And this was confirmed by docking experiments. We also assessed the substrates range of BphAELB400 and its variants with degradation activity. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the 3-6 chlorinated biphenyls, which are generally very poorly oxidized by most dioxygenases. Collectively, the present work showed a significant effect of mutation Ser283Met on substrate specificity/regiospecificity in BPDO. It will certainly be meaningful elements for understanding the effect of the residue corresponding to 283 in other Rieske oxygenase enzymes. Importance The segment 280-283 in BphAEs is located at the entrance of the catalytic pocket and it shows variation conformation. In previous works, the results have been suggested but never proved residue Ser283 of BphAELB400 might play a role in substrate specificity. In the present paper, we found the Ser283Met substitution significantly increased the specificity of the reaction of BphAE toward biphenyl, 2,3′,4,4′-CB, 2,2′,6,6′-CB and 2,3′,4,4′,5-CB. Meanwhile, the Ser283Met substitution altered regiospecificity of BphAE toward 2,2′-dichlorobiphenyl and 2,6-dichlorobiphenyl. Additionally, this substitution extended the range of PCBs metabolized by the mutated BphAE. BphAES283M and BphAEp4-S283M were clearly improved in oxidizing some of the more highly chlorinated biphenyls (3-6 Chlorines; Table 4), which are generally very poorly oxidized by most dioxygenases. We used modeled and docked enzymes to identify some of the structural features that explain the new properties of the mutant enzymes. Altogether, this study provides better insights into the mechanisms by which BPDO evolves to change and/or expand its substrate range and its regiospecificity.