Substrate specificity of Sphingobium chlorophenolicum 2,6-dichlorohydroquinone 1,2-dioxygenase

Biochemistry. 2011 Oct 18;50(41):8899-913. doi: 10.1021/bi200855m. Epub 2011 Sep 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alphaproteobacteria / enzymology*
  • Catalysis
  • Chemistry / methods
  • Dioxygenases / chemistry*
  • Halogens
  • Hydroquinones / chemistry
  • Kinetics
  • Ligands
  • Magnetic Resonance Spectroscopy / methods
  • Models, Chemical
  • Oxygen / chemistry
  • Phenols / chemistry
  • Substrate Specificity

Substances

  • Halogens
  • Hydroquinones
  • Ligands
  • Phenols
  • 2,6-dichlorohydroquinone dioxygenase
  • Dioxygenases
  • Oxygen
  • hydroquinone