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Int J Biol Sci 2011; 7(8):1171-1179. doi:10.7150/ijbs.7.1171

Research Paper

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

Wanpeng Sun1, Ramaswami Sammynaiken2, 3, Lifeng Chen1, Jason Maley2, Gabriele Schatte2, Yijiang Zhou4, Jian Yang1,✉

1. College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada
2. Saskatchewan Structural Sciences Centre, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada
3. Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada
4. School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310058, P.R. of China

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 kcat and Km 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.

Keywords: Alkaline resistance, thermal stability, radical intermediate, EPR, biodegradation

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How to cite this article:
Sun W, Sammynaiken R, Chen L, Maley J, Schatte G, Zhou Y, Yang J. Sphingobium Chlorophenolicum Dichlorohydroquinone Dioxygenase (PcpA) Is Alkaline Resistant and Thermally Stable. Int J Biol Sci 2011; 7(8):1171-1179. doi:10.7150/ijbs.7.1171. Available from http://www.ijbs.com/v07p1171.htm