Cloning, overexpression, purification, and characterization of the maleylacetate reductase from Sphingobium chlorophenolicum strain ATCC 53874.
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.
Chen
Lifeng
L
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.
Maloney
Katharine
K
Krol
Ed
E
Zhu
Bin
B
Yang
Jian
J
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
D001426:Bacterial Proteins; D010416:Pentachlorophenol; D044925:Oxidoreductases Acting on CH-CH Group Donors; C021530:maleylacetate reductase