title: Cloning, overexpression, purification, and characterization of the maleylacetate reductase from Sphingobium chlorophenolicum strain ATCC 53874.
abstract: The final enzyme in the pentachlorophenol (PCP) biodegradation pathway in Sphingobium chlorophenolicum is maleylacetate reductase (PcpE), which catalyzes the reductive dehalogenation of 2-chloromaleylacetate to maleylacetate and the subsequent reduction of malyelacetate to 3-oxoadipate. In this study, the pcpE gene was cloned from S. chlorophenolicum strain ATCC 53874 and overexpressed in Escherichia coli BL21-AI cells. The recombinant PcpE, purified to higher than 95% purity using affinity chromatography, exhibited optimal activity at pH 7.0. The kinetic parameters k(cat) and K(m) were 1.2 +/- 0.3 s(-1) and 0.09 +/- 0.04 mM, respectively, against maleylacetate under the optimal pH. In addition, the purified PcpE was able to restore PCP-degrading capability to S. chlorophenolicum strain ATCC 39723, implicating that there was no functional PcpE in the ATCC 39723 strain.
authors.affiliation: College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Chen
authors.forename: Katharine
authors.initials: K
authors.lastname: Maloney
authors.forename: Ed
authors.initials: E
authors.lastname: Krol
authors.forename: Bin
authors.initials: B
authors.lastname: Zhu
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010416:Pentachlorophenol / Q000378:metabolism; D042301:Sphingomonadaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010416:Pentachlorophenol; D044925:Oxidoreductases Acting on CH-CH Group Donors; C021530:maleylacetate reductase