Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus (original) (raw)

2003, Applied and Environmental Microbiology

Updated information and services can be found at: These include: REFERENCES http://aem.asm.org/content/69/8/4628#ref-list-1 at: This article cites 46 articles, 12 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ؎ 1 kDa, whereas the native molecular mass was 71 ؎ 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-␤-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 ؎ 0.1 mol of cobalamin, 0.6 ؎ 0.02 mol of cobalt, 7.1 ؎ 0.6 mol of iron, and 5.8 ؎ 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 ؎ 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K m values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 ؎ 3.2, 23.7 ؎ 5.2, and 47 ؎ 10 M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced.