Isomer-specific 3-chloroacrylic acid dehalogenases function in the bacterial degradation of 1,3-dichloropropene, a compound used in agriculture to kill plant-parasitic nematodes. The crystal structure of the heterohexameric trans-3-chloroacrylic acid dehalogenase (CaaD) from Pseudomonas pavonaceae 170 inactivated by 3-bromopropiolate shows that Glu-52 in the ␣-subunit is positioned to function as the water-activating base for the addition of a hydroxyl group to C-3 of 3-chloroacrylate and 3-bromopropiolate, whereas the nearby Pro-1 in the ␤-subunit is positioned to provide a proton to C-2. Two arginine residues, ␣Arg-8 and ␣Arg-11, interact with the C-1 carboxylate groups, thereby polarizing the ␣,␤-unsaturated acids. The reaction with 3-chloroacrylate results in the production of an unstable halohydrin, 3-chloro-3-hydroxypropanoate, which decomposes into the products malonate semialdehyde and HCl. In the inactivation mechanism, however, malonyl bromide is produced, which irreversibly alkylates the ␤Pro-1. CaaD is related to 4-oxalocrotonate tautomerase, with which it shares an N-terminal proline. However, in 4-oxalocrotonate tautomerase, Pro-1 functions as a base participating in proton transfer within a hydrophobic active site, whereas in CaaD, the acidic proline is stabilized in a hydrophilic active site. The altered active site environment of CaaD thus facilitates a previously unknown reaction in the tautomerase superfamily, the hydration of the ␣,␤-unsaturated bonds of trans-3-chloroacrylate and 3-bromopropiolate. The mechanism for these hydration reactions represents a novel catalytic strategy that results in carbon-halogen bond cleavage.Dehalogenases are enzymes that cleave carbon-halogen bonds. They are found in various bacteria, allowing them to use halogenated hydrocarbons as growth substrates (1, 2). Detailed three-dimensional structural information is available for dehalogenases such as haloalkane dehalogenase, 2-haloacid dehalogenase, and haloalcohol dehalogenase, which catalyze the cofactor-independent cleavage of the covalent bond between a halogen and an sp 3 -hybridized carbon atom by substitution mechanisms (3-5). In addition, several cofactor-dependent dehalogenases have been characterized that cleave the bond between a halogen and an sp 2 -hybridized carbon atom. Examples include heme-dependent reductive dehalogenases (6, 7) and the 4-chlorobenzoyl-CoA dehalogenases (8). In contrast, cofactorindependent dehalogenases that cleave the bond between a halogen and an sp 2 -hybridized carbon atom have only recently been discovered (9, 10).The 3-chloroacrylic acid dehalogenases from Pseudomonas pavonaceae 170 represent cofactor-independent dehalogenases that catalyze the cleavage of vinylic carbon-halogen bonds, in which the halogen is bound to an sp 2 -hybridized carbon atom (9 -11). They are part of a multienzyme degradation route for the cis-and trans-isomers of 1,3-dichloropropene (DCP).1 Cisand trans-DCP are components of the commercially produced fumigants Shell D-D and Telone II, which are us...

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