Determination of the active site of Sphingobium chlorophenolicum 2,6-dichlorohydroquinone dioxygenase (PcpA)

Abstract

2,6-Dichlorohydroquinone 1,2-dioxygenase (PcpA) from Sphingobium chlorophenolicum ATCC 39723 is a member of a class of Fe(II)-containing hydroquinone dioxygenases that is involved in the mineralization of the pollutant pentachlorophenol. This enzyme has not been extensively characterized, despite its interesting ring-cleaving activity and use of Fe(II), which are reminiscent of the well-known extradiol catechol dioxygenases. On the basis of limited sequence homology to the extradiol catechol dioxygenases, the residues ligating the Fe(II) center were originally proposed to be H159, H227, and E276 (Xu et al. in Biochemistry 38:7659-7669, 1999). However, PcpA has higher sequence homology to a newly reported, crystallographically characterized zinc metalloenzyme that has a similar predicted fold. We generated a homology model of the structure of PcpA based upon the structure of this zinc metalloenzyme. The homology model predicts that the tertiary structure of PcpA differs significantly from that of the extradiol dioxygenases, and that the residues ligating the Fe(II) are H11, H227, and E276. This structural model was tested by mutating each of H11, H159, H227, and E276 to alanine. An additional residue that is predicted to lie near the active site and is conserved among PcpA, its closest homologues, and the extradiol dioxygenases, Y266, was mutated to phenylalanine. Of these mutants, only H159A retained significant activity, thus confirming the active-site location predicted by the homology-based structural model. The model provides an important basis for understanding the origin of the unique function of PcpA.

Catabolism of Phenol and Its Derivatives in Bacteria

“…HDs of bacteria, containing Fe 2þ cations, have been classified into two different classes: monomeric or consisting of two different subunits. Monomeric 2,6-dichlorohydroquinone 1,2-dioxygenase, homologous to C2,3D and catalyzing the oxidative cleavage of 2,6-dichlorohydroquinone in the PCP degrader Sphingobium chlorophenolicum, was characterized in detail (Machonkin & Doerner, 2011). An HD composed of two different subunits and oxidizing a wide range of hydroquinones to the corresponding 4-hydroxymuconate semialdehydes was isolated from the alkylphenol-degrading bacterium Sphingomonas sp.…”

Sphingobium Chlorophenolicum Dichlorohydroquinone Dioxygenase (PcpA) Is Alkaline Resistant and Thermally Stable

“…It would be interesting to see whether these mutations affect the pH and temperature tolerances for PcpA. In addition, a structural model of PcpA indicated the presence of three histidine and one glutamate residues at the catalytic active site 33, suggesting the ionization state of these residues may also play an important role in determining PcpA enzyme activity.…”

“…Because 2,6-DCHQ is a common metabolic intermediate of several chloroaromatic compounds, ring cleavage and dehalogenation catalyzed by PcpA are key steps involved in degradation of recalcitrant chlorophenols 31-33. Unlike the intradiol, extradiol and gentisate dioxygenases, PcpA catalyzes the cleavage of aromatic rings between a hydroxyl group and a chlorine group 29-31, 33. Sequence analysis suggests that PcpA belongs to the vicinal oxygen chelate (VOC) protein superfamily 33.…”

“…Unlike the intradiol, extradiol and gentisate dioxygenases, PcpA catalyzes the cleavage of aromatic rings between a hydroxyl group and a chlorine group 29-31, 33. Sequence analysis suggests that PcpA belongs to the vicinal oxygen chelate (VOC) protein superfamily 33. Although its predicted tertiary structure differs significantly from that of the extradiol dioxygenases, PcpA may follow a catalytic mechanism similar to that of the extradiol dioxygenases 33.…”

“…Sequence analysis suggests that PcpA belongs to the vicinal oxygen chelate (VOC) protein superfamily 33. Although its predicted tertiary structure differs significantly from that of the extradiol dioxygenases, PcpA may follow a catalytic mechanism similar to that of the extradiol dioxygenases 33.…”