trans-3-Chloroacrylic acid dehalogenase from Pseudomonas pavonaceae 170 shares structural and mechanistic similarities with 4-oxalocrotonate tautomerase.
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.
Poelarends
G J
GJ
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
Saunier
R
R
Janssen
D B
DB
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D006867:Hydrolases; C077721:trans-3-chloroacrylic acid dehalogenase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases