title: 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.
authors.affiliation: Department of Chemistry, Whitman College, 345 Boyer Ave., Walla Walla, WA 99362, USA. machonte@whitman.edu
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Machonkin
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Holland
authors.forename: Kristine N
authors.initials: KN
authors.lastname: Smith
authors.forename: Justin S
authors.initials: JS
authors.lastname: Liberman
authors.forename: Adriana
authors.initials: A
authors.lastname: Dinescu
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Cundari
authors.forename: Sara S
authors.initials: SS
authors.lastname: Rocks
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D042301:Sphingomonadaceae / Q000201:enzymology*
chemical_list: C119974:2,6-dichlorohydroquinone dioxygenase; D049308:Dioxygenases