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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.

SUBMITTER: Poelarends GJ

PROVIDER: S-EPMC95317 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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

Poelarends G J GJ Saunier R R Janssen D B DB

Journal of bacteriology 20010701 14

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 turnove ...[more]

PMID: 11418568

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Project description:4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

Project description:A 149-amino acid protein designated Cg10062 is encoded by a gene from Corynebacterium glutamicum. The physiological function of Cg10062 is unknown, and the gene encoding this protein has no obvious genomic context. Sequence analysis links Cg10062 to the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) family, one of the five known families of the tautomerase superfamily. The characterized tautomerase superfamily members have two distinctive characteristics: a beta-alpha-beta structure motif and a catalytic amino-terminal proline. Pro-1 is present in the Cg10062 amino acid sequence along with His-28, Arg-70, Arg-73, Tyr-103, and Glu-114, all of which have been implicated as critical residues for cis-CaaD activity. The gene for Cg10062 has been cloned and the protein overproduced, purified, and subjected to kinetic and mechanistic characterization. Like cis-CaaD, Cg10062 functions as a hydratase: it converts 2-oxo-3-pentynoate to acetopyruvate and processes 3-bromopropiolate to a species that inactivates the enzyme by acylation of Pro-1. Kinetic and (1)H NMR spectroscopic studies also show that Cg10062 processes both isomers of 3-chloroacrylic acid at low levels with a clear preference for the cis isomer. Pro-1 is critical for the dehalogenase and hydratase activities because the P1A mutant no longer catalyzes either reaction. The presence of the six key catalytic residues and the hydratase activity coupled with the absence of an efficient cis-CaaD activity and the lack of isomer specificity implicate factors beyond this core set of residues in cis-CaaD catalysis and specificity. This work sets the stage for in-depth mechanistic and structural studies of Cg10062, which could identify the additional features necessary for a fully active and highly specific cis-CaaD. Such results will also shed light on how cis-CaaD emerged in the tautomerase superfamily because Cg10062 could be characteristic of an intermediate along the evolutionary pathway for this dehalogenase.

Dataset Information

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

Publications

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

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