Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans.

Julya Krasotkina,Keith A Maruya,Tina Walters,Stephen W Ragsdale

doi:10.1074/jbc.m106217200

Julya Krasotkina, Keith A Maruya + Show 2 more

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https://doi.org/10.1074/jbc.m106217200

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Abstract

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.

Highlights

Associated with the United Nations Environmental Program, in May 2001, a global treaty has been adopted that aims to eliminate 12 persistent organic pollutants, the so-called “dirty dozen,” which are chemicals that pose a significant risk to health and the environment
Are organocobalt adducts analogous to those in B12-dependent methyltransferases formed during the reaction mechanism? How is the chlorine replaced by a hydride equivalent? Does the reaction involve radical intermediates? What are the structural requirements for the substrate of the dehalogenase reaction? Here we describe studies focused mainly on characterizing the substrate specificity and mechanism of the aromatic dehalogenase from Desulfitobacterium chlororespirans, an organism that was enriched and isolated based on its ability to grow on 3-chloro-4-hydroxybenzoate [17]
Purification of CprA—The 3-chloro-4-hydroxybenzoate reductive dehalogenase (CprA) was purified from D. chlororespirans cells that were grown on pyruvate as a carbon and electron source and 3-chloro-4-hydroxybenzoate as an electron acceptor

Summary

EXPERIMENTAL PROCEDURES

Organism and Growth Conditions—D. chlororespirans (ATCC 700175) was grown with agitation on reduced anaerobic medium (ATCC 2035), containing 20 mM pyruvate, 2 mM 3-chloro-4-hydroxybenzoate, and 1 g of yeast extract per liter [17]. TLC—The assay mixture, which contained 0.3 ml of buffer B (pH 7.6), 40 ␮l of 15 mM methyl viologen, 50 ␮l of 0.2 M titanium citrate, 10 ␮l of 50 mM 3-chloro-4-hydroxybenzoate, and 10 –100 ␮l of sample, was anaerobically incubated for 30 min at 57 °C and quenched by adding 50 ␮l of 0.2 M perchloric acid and centrifuged at 10,000 ϫ g for 1 min. Spectrophotometric Dehalogenase Assay and Determination of Kinetic Parameters—Dehalogenation of 3-chloro-4-hydroxybenzoate and other chlorinated substrates was measured spectrophotometrically at 578 nm and 37 °C by following the oxidation of reduced methyl viologen (⑀578 ϭ 9.7 mMϪ1 cmϪ1) [19]. The organic phase was analyzed for the parent compound (3,3Ј,5,5Ј-tetrachloro-4,4Ј-biphenyldiol) and dechlorination products by gas chromatography-mass spectrometry (GC-MS). Blotting was carried out at 36 V for 1.5 h using a transfer

RESULTS AND DISCUSSION

Vmax kcat

TABLE III Inhibition of dehalogenase activity by chlorinated hydrocarbons