title: Purification, cloning, and sequencing of an enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from Clostridium bifermentans DPH-1.
abstract: An enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from cell-free extracts of Clostridium bifermentans DPH-1 was purified, cloned, and sequenced. The enzyme catalyzed the reductive dechlorination of PCE to cis-1,2-dichloroethylene via trichloroethylene, at a Vmax and Km of 73 nmol/mg protein and 12 microM, respectively. Maximal activity was recorded at 35 degrees C and pH 7.5. Enzymatic activity was independent of metal ions but was oxygen sensitive. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. The molecular mass of the native enzyme was estimated to be approximately 70 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) revealed molecular masses of approximately 35 kDa and 35.7 kDa, respectively. A broad spectrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropane, and 1,1,2-trichloroethane) was degraded. With degenerate primers designed from the N-terminal sequence (27 amino acid residues), a partial sequence (81 bp) of the encoding gene was amplified by polymerase chain reaction (PCR) and sequenced. Southern analysis of C. bifermentans genomic DNA using the PCR product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase.
authors.affiliation: Department of Bioprocessing, Gifu University, Japan.
authors.forename: B C
authors.initials: BC
authors.lastname: Okeke
authors.forename: Y C
authors.initials: YC
authors.lastname: Chang
authors.forename: M
authors.initials: M
authors.lastname: Hatsu
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: K
authors.initials: K
authors.lastname: Takamizawa
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013750:Tetrachloroethylene / Q000378:metabolism*
chemical_list: D010088:Oxidoreductases; C112990:tetrachloroethene dehalogenase; D013750:Tetrachloroethylene