title: Substrate specificity of Sphingobium chlorophenolicum 2,6-dichlorohydroquinone 1,2-dioxygenase.
abstract: PcpA is an aromatic ring-cleaving dioxygenase that is homologous to the well-characterized Fe(II)-dependent catechol extradiol dioxygenases. This enzyme catalyzes the oxidative cleavage of 2,6-dichlorohydroquinone in the catabolism of pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. (1)H NMR and steady-state kinetics were used to determine the regiospecificity of ring cleavage and the substrate specificity of the enzyme. PcpA exhibits a high degree of substrate specificity for 2,6-disubstituted hydroquinones, with halogens greatly preferred at those positions. Notably, the k(cat)(app)/K(mA)(app) of 2,6-dichlorohydroquinone is ~40-fold higher than that of 2,6-dimethylhydroquinone. The asymmetric substrate 2-chloro-6-methylhydroquinone yields a mixture of 1,2- and 1,6-cleavage products. These two modes of cleavage have different K(mO(2))(app) values (21 and 260 μM, respectively), consistent with a mechanism in which the substrate binds in two catalytically productive orientations. In contrast, monosubstituted hydroquinones show a limited amount of ring cleavage but rapidly inactivate the enzyme in an O(2)-dependent fashion, suggesting that oxidation of the Fe(II) may be the cause. Potent inhibitors of PcpA include ortho-disubstituted phenols and 3-bromocatechol. 2,6-Dibromophenol is the strongest competitive inhibitor, consistent with PcpA's substrate specificity. Several factors that could yield this specificity for halogen substituents are discussed. Interestingly, 3-bromocatechol also inactivates the enzyme, while 2,6-dihalophenols do not, indicating a requirement for two hydroxyl groups for ring cleavage and for enzyme inactivation. These results provide mechanistic insights into the hydroquinone dioxygenases.
authors.affiliation: Department of Chemistry, Whitman College, 345 Boyer Avenue, Walla Walla, Washington 99362, USA. machonte@whitman.edu
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Machonkin
authors.forename: Amy E
authors.initials: AE
authors.lastname: Doerner
mesh_terms: D020561:Alphaproteobacteria / Q000201:enzymology*; D002384:Catalysis; D002621:Chemistry / Q000379:methods; D049308:Dioxygenases / Q000737:chemistry*; D006219:Halogens; D006873:Hydroquinones / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry; D010636:Phenols / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D006219:Halogens; D006873:Hydroquinones; D008024:Ligands; D010636:Phenols; C119974:2,6-dichlorohydroquinone dioxygenase; D049308:Dioxygenases; D010100:Oxygen; C031927:hydroquinone