Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans

Abstract

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 V(max) of 28 units/mg protein and a K(m) of 20 μM. The pH and temperature optimum were 8.2 and 52 °C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (E(m)) = - 440 mV), one [3Fe-4S] cluster (E(m) = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an E(m) 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.