Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans.
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.
Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. Bran,vandepas@algemeen.micr.wau.nl
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van de Pas
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Smidt
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Hagen
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van der Oost
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Schraa
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Stams
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de Vos
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000502:physiology; D001483:Base Sequence; D004269:DNA, Bacterial; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000502:physiology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D014805:Vitamin B 12 / Q000378:metabolism
D004269:DNA, Bacterial; D010088:Oxidoreductases; C120588:ortho-chlorophenol reductive dehalogenase; D014805:Vitamin B 12