title: Sphingobium chlorophenolicum dichlorohydroquinone dioxygenase (PcpA) is alkaline resistant and thermally stable.
abstract: Dichlorohydroquinone dioxygenase (PcpA) is the ring-cleavage enzyme in the PCP biodegradation pathway in Sphingobium chlorophenolicum strain ATCC 39723. PcpA dehalogenates and oxidizes 2,6-dichlorohydroquinone to form 2-chloromaleylacetate, which is subsequently converted to succinyl coenzyme A and acetyl coenzyme A via 3-oxoadipate. Previous studies have shown that PcpA is highly substrate-specific and only uses 2,6-dichlorohydroquinone as its substrate. In the current study, we overexpressed and purified recombinant PcpA and showed that PcpA was highly alkaline resistant and thermally stable. PcpA exhibited two activity peaks at pH 7.0 and 10.0, respectively. The apparent k(cat) and K(m) were measured as 0.19 ± 0.01 s(-1) and 0.24 ± 0.08 mM, respectively at pH 7.0, and 0.17 ± 0.01 s(-1) and 0.77 ± 0.29 mM, respectively at pH 10.0. Electron paramagnetic resonance studies showed rapid oxidation of Fe(II) to Fe(III) in PcpA and the formation of a stable radical intermediate during the enzyme catalysis. The stable radical was predicted to be an epoxide type dichloro radical with the unpaired electron density localized on C3.
authors: College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
authors: Wanpeng
authors: W
authors: Sun
authors: Ramaswami
authors: R
authors: Sammynaiken
authors: Lifeng
authors: L
authors: Chen
authors: Jason
authors: J
authors: Maley
authors: Gabriele
authors: G
authors: Schatte
authors: Yijiang
authors: Y
authors: Zhou
authors: Jian
authors: J
authors: Yang
mesh_terms: D001673:Biodegradation, Environmental; D002942:Circular Dichroism; D049308:Dioxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010416:Pentachlorophenol / Q000378:metabolism*; D042301:Sphingomonadaceae / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D010416:Pentachlorophenol; D007501:Iron; C119974:2,6-dichlorohydroquinone dioxygenase; D049308:Dioxygenases