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Journal of Bacteriology, July 2001, p. 4269-4277, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4269-4277.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

trans-3-Chloroacrylic Acid Dehalogenase from Pseudomonas pavonaceae 170 Shares Structural and Mechanistic Similarities with 4-Oxalocrotonate Tautomerase

Gerrit J. Poelarends,dagger Raymond Saunier, and Dick B. Janssen*

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands

Received 16 January 2001/Accepted 18 April 2001

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.

* Corresponding author. Mailing address: Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Phone: 31-50-3634209. Fax: 31-50-3634165. E-mail: d.b.janssen{at}chem.rug.nl.

dagger Present address: Department of Microbiology, University of Groningen, 9751 NN Haren, The Netherlands.

Journal of Bacteriology, July 2001, p. 4269-4277, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4269-4277.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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