Document Detail

Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans.
MedLine Citation:
PMID:  10400648     Owner:  NLM     Status:  MEDLINE    
ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.
B A van de Pas; H Smidt; W R Hagen; J van der Oost; G Schraa; A J Stams; W M de Vos
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  274     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1999 Jul 
Date Detail:
Created Date:  1999-08-19     Completed Date:  1999-08-19     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  20287-92     Citation Subset:  IM    
Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. Bran,
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MeSH Terms
Amino Acid Sequence
Bacteria, Anaerobic / enzymology*,  physiology
Base Sequence
DNA, Bacterial
Electron Spin Resonance Spectroscopy
Electron Transport
Gram-Positive Bacteria / enzymology*,  physiology
Molecular Sequence Data
Oxidoreductases / chemistry,  genetics,  isolation & purification*
Sequence Homology, Amino Acid
Vitamin B 12 / metabolism
Reg. No./Substance:
0/DNA, Bacterial; 68-19-9/Vitamin B 12; EC 1.-/Oxidoreductases; EC 1.97.-/ortho-chlorophenol reductive dehalogenase

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