The genes (caaD1 and caaD2) encoding the trans-3-chloroacrylic acid dehalogenase (CaaD) of the 1,3-dichloropropene-utilizing bacterium Pseudomonas pavonaceae 170 were cloned and heterologously expressed in Escherichia coli and Pseudomonas sp. strain GJ1. CaaD is a protein of 50 kDa that is composed of ␣-subunits of 75 amino acid residues and ␤-subunits of 70 residues. It catalyzes the hydrolytic cleavage of the ␤-vinylic carbonchlorine bond in trans-3-chloroacrylic acid with a turnover number of 6.4 s ؊1. On the basis of sequence similarity, oligomeric structure, and subunit size, CaaD appears to be related to 4-oxalocrotonate tautomerase (4-OT). This tautomerase consists of six identical subunits of 62 amino acid residues and catalyzes the isomerization of 2-oxo-4-hexene-1,6-dioate, via hydroxymuconate, to yield 2-oxo-3-hexene-1,6-dioate. In view of the oligomeric architecture of 4-OT, a trimer of homodimers, CaaD is postulated to be a hexameric protein that functions as a trimer of ␣␤-dimers. The sequence conservation between CaaD and 4-OT and site-directed mutagenesis experiments suggested that Pro-1 of the ␤-subunit and Arg-11 of the ␣-subunit are active-site residues in CaaD. Pro-1 could act as the proton acceptor/donor, and Arg-11 is probably involved in carboxylate binding. Based on these findings, a novel dehalogenation mechanism is proposed for the CaaD-catalyzed reaction which does not involve the formation of a covalent enzyme-substrate intermediate.Isomer-specific 3-chloroacrylic acid dehalogenases catalyze the hydrolytic cleavage of the ␤-vinylic carbon-chlorine bond in either cis-or trans-3-chloroacrylic acid to yield malonic acid semialdehyde and HCl. These enzymes are produced by both gram-positive and gram-negative bacteria, including Pseudomonas pavonaceae 170 (27), Pseudomonas cepacia CAA1 (11), and the coryneform bacterial strains FG41 (47) and CAA2 (11), enabling these organisms to use one or both isomers of the xenobiotic compound 3-chloroacrylic acid for growth. The dehalogenases from strain FG41 were purified to homogeneity, and trans-3-chloroacrylic acid dehalogenase (CaaD) was found to be a 50-kDa enzyme composed of different subunits of 8.7 and 7.4 kDa, whereas the cis-3-chloroacrylic acid dehalogenase was an enzyme composed of two or three identical 16-kDa subunits (47). Although large fragments of these dehalogenating enzymes were sequenced, no significant sequence similarities with other protein sequences were found when the different databases were searched in 1992 (47).Whereas most hydrolytic dehalogenase that are active with halogenated aliphatic compounds (so-called halidohydrolases), such as haloalkane dehalogenases (26, 30, 50), haloacetate dehalogenases (15-17), and 2-haloacid dehalogenases (21, 25, 31), are only able to displace halogens bound to sp 3 -hybridized carbon atoms, 3-chloroacrylic acid dehalogenases are unique in that they can cleave the much more stable vinylic carbon-halogen bond, in which the halogen is bound to an sp 2 -hybridized carbon atom. Cleavage of the...