Purification, cloning, and sequencing of an enzyme mediating the reductivedechlorination of tetrachloroethylene (PCE) from Clostridium bifermentans DPH-1

An enzyme mediating the reductive dechlorination of tetrachloroethylene (PC E) 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 V-max and K-m of 73 nmol/mg protein and 12 muM, respectively. Maximal activity was record ed at 35 degreesC and pH 7.5. Enzymatic activity was independent of metal i ons but was oxygen sensitive. A mixture of propyl iodide and titanium citra te caused a light-reversible inhibition of enzymatic activity suggesting th e involvement of a corrinoid cofactor. The molecular mass of the native enz yme was estimated to be approximately 70 kDa. Sodium dodecyl sulfate-polyac rylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorptio n ionization-time of flight/mass spectrometry (MALDI-TOF/MS) revealed molec ular masses of approximately 35 kDa and 35.7 kDa, respectively. A broad spe ctrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-d ichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dich loropropane, and 1,1,2-trichloroethane) was degraded. With degenerate prime rs designed from the N-terminal sequence (27 amino acid residues), a partia l sequence (81 bp) of the encoding gene was amplified by polymerase chain r eaction (PCR) and sequenced. Southern analysis of C. bifermentans genomic D NA using the PCR product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was clo ned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no hom ology with any known dehalogenase, suggesting a distinct PCE dehalogenase.