trans-3-Chloroacrylic acid dehalogenase from Pseudomonas pavonaceae 170 shares structural and mechanistic similarities with 4-oxalocrotonate tautomerase

Abstract

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 alpha-subunits of 75 amino acid residues and beta-subunits of 70 residues. It catalyzes the hydrolytic cleavage of the beta-vinylic carbon-chlorine 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 alpha beta-dimers. The sequence conservation between CaaD and 4-OT and site-directed mutagenesis experiments suggested that Pro-1 of the beta-subunit and Arg-11 of the alpha-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.

FIG. 1

Alignments of the amino acid sequences of the α-subunit (CaaD1) and β-subunit (CaaD2) of CaaD with the seven 4-OT sequences (A and B) and with the amino-terminal sequences of the subunits of the CaaD isolated from strain FG41 (C). Residues conserved throughout all sequences are indicated by an asterisk. Dashes represent residues absent in other sequences. The catalytically important residues in 4-OT are marked with + and shown in boldface. The region of highest sequence identity among CaaD1 and the 4-OT sequences is boxed. Ppa, P. pavonaceae; Bs, B. subtilis; Psp, Pseudomonas sp.; Pp, P. putida; Ps, P. stutzeri; Sa, S. aromaticivorans; Csp, coryneform bacterial strain.

FIG. 2

Comparative reaction scheme for 4-OT and CaaD. The primed residues come from other subunits. (A) Reaction catalyzed by 4-OT (adopted from references and 41). (B) Two proposed reaction schemes for CaaD, one involving the formation of 3-chloro-3-hydroxypropanoic acid (route 1) and the other involving the formation of a carbanion intermediate (route 2). Pro-1 is shown as the catalytic base to activate a water molecule that attacks the substrate. Arg-11 is implicated in substrate binding.