Crystal Structures of Native and Inactivated cis-3-Chloroacrylic Acid Dehalogenase

Abstract

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis-and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro The cis-and trans-3-chloroacrylic acid dehalogenases (cisCaaD and CaaD) 4 catalyze the cofactor-independent hydrolytic dehalogenation of, respectively, the cis-and trans-isomers of 3-chloroacrylic acid (1 and 2, Scheme 1) to produce malonate semialdehyde (5) and HCl (1-3). Both reactions may be initiated by the attack of water at C3 to form an enzyme-stabilized enediolate intermediate (3). Subsequent ketonization of 3 with protonation at C2 generates a chlorohydrin intermediate (4), which can collapse by direct expulsion of the chloride to afford 5 (1, 4, 5). Alternatively, ketonization of 3 can result in chloride loss and the formation of the enol intermediate, 6, which tautomerizes to afford 5. The two enzymes are found in bacterial pathways that convert the cis-and trans-isomers of 1,3-dichloropropene, used as nematocides, to acetaldehyde (7) and carbon dioxide (6).The cis-and trans-3-chloroacrylic acid dehalogenases have low sequence identity (ϳ20%) and different oligomerization states (1-3). CaaD is a heterohexamer consisting of three 75-residue ␣-chains and three 70-residue ␤-chains, whereas cis-CaaD forms a homotrimer of three identical 149-residue polypeptide chains, which can be considered as the fusion product of a CaaD ␣-and ␤-chain (2, 3, 5). As a result, the two enzymes have been classified in two different families in the tautomerase superfamily, with each being related to 4-oxalocrotonate tautomerase (7-9). Yet, the differences in catalytic efficiency are only modest, and major elements of the catalytic mechanisms are conserved. In both, a glutamate residue (Glu-114 in cis-CaaD and ␣Glu-52 in CaaD) is proposed to function as a general base catalyst to activate a water molecule for attack at C3 (of 1 or 2) and the N-terminal proline (Pro-1 in cis-CaaD and ␤Pro-1 in CaaD) is believed to provide a proton at C2. Two * This work was supported in part by United States Public Health Services Grant GM 65324. The mass spectrometry described in this paper was carried out in the Analytical Instrumentation Facility Core housed in the College of Pharmacy at the University of Texas at Austin and supported by Center Grant ES07784. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Sectio...

Reactions of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue, with Acetylene and Allene Substrates: Evidence for a Hydration-Dependent Decarboxylation

“…24,25 Covalent modification resulted from the incubation of cis -CaaD with the ( R )-isomer of oxirane-2-carboxylate (13). 24 Mutagenesis confirmed the importance of these two residues for cis -CaaD activity.…”

“…In this mechanism, the carboxylate group interacts with the three positively charged residues (Arg-70, Arg-73, and His-28). 25,27 The interactions serve to bind and polarize the substrate. Polarization of the substrate generates a partial positive charge at C-3 and sets up the substrate for a Michael addition of water.…”

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

“…Haloalkane dehalogenase, haloacid dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase all use an aspartate nucleophile and hydrolyze the intermediate enzyme-substrate ester to catalyze an overall hydrolytic displacement reaction (15). cis-3-Chloroacrylic acid dehalogenase proceeds via a hydratase-like mechanism (16). These structurally defined dehalogenases, which use water as a cosubstrate, are not metalloenzymes.…”

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

“…Other dehalogenases, such as corrinoid/iron-sulfur cluster-containing reductive dehalogenases 5,50) and cis-and trans-3-chloroacrylic acid dehalogenases, 51,52) are known to act on unsaturated organohalogen compounds, but information on the mechanism of enzymatic degradation of such compounds is limited. To get insight into the enzymatic dehalogenation of unsaturated organohalogen compounds, we searched for microorganisms that grow on 2-chloroacrylate (2-CAA) as sole source of carbon and energy.…”

“…Lactate-grown 2-Haloacrylate hydratase resembles cis-and trans-3-chloroacrylic acid dehalogenases 51,52) in that it catalyzes the dehalogenation of the substrate by the addition of a water molecule to the carbon-carbon double bond, but it is significantly different from 3-chloroacrylic acid dehalogenases not only in its substrate specificity and primary structure but also in its cofactor requirement. No cofactor is required for 3-chloroacrylic acid dehalogenases, whereas FADH 2 is an absolute requirement for the 2-haloacrylate hydratase reaction.…”