Binding modes of DL-2-haloacid dehalogenase revealed by crystallography, modeling and isotope effects studies.

Several pathways of biotic dechlorination can be found in enzymes, each characterized by different chlorine isotopic fractionation, which can thus serve as a signature of a particular mechanism. Unlike other dehalogenases, DL-2-haloacid dehalogenase, DL-DEX, converts both enantiomers of the substrate. Chlorine isotope effects for this enzyme are larger than in the case of other dehalogenases. Recently, the 3D structure of this enzyme became available and enabled us to model these isotope effects and seek their origin. We show that the elevated values of the chlorine kinetic isotope effects originate in part in the processes of binding and migration within the enzyme active site that precede the dehalogenation step.

Substrate specificity of fluoroacetate dehalogenase: an insight from crystallographic analysis, fluorescence spectroscopy, and theoretical computations.

The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 Å resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.

Crystallization and preliminary X-ray analysis of L-azetidine-2-carboxylate hydrolase from Pseudomonas sp. strain A2C.

L-Azetidine-2-carboxylate hydrolase from Pseudomonas sp. strain A2C catalyzes a ring-opening reaction that detoxifies L-azetidine-2-carboxylate, an analogue of L-proline. Recombinant L-azetidine-2-carboxylate hydrolase was overexpressed, purified and crystallized using polyethylene glycol and magnesium acetate as precipitants. The needle-shaped crystal belonged to space group P2(1), with unit-cell parameters a = 35.6, b = 63.6, c = 54.7 A, beta = 105.5 degrees . The crystal diffracted to a resolution of 1.38 A. The calculated V(M) value was 2.2 A(3) Da(-1), suggesting that the crystal contains one enzyme subunit in the asymmetric unit.

The catalytic mechanism of fluoroacetate dehalogenase: a computational exploration of biological dehalogenation.

The biological dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole-enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen-bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the alpha-carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C-F bond is greatly facilitated by the hydrogen-bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.

X-Ray crystallographic and mutational studies of fluoroacetate dehalogenase from Burkholderia sp. strain FA1.

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the alpha/beta hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the N(epsilon1) atom of Trp150 and the N(epsilon2) atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.

Expression, purification and preliminary X-ray characterization of DL-2-haloacid dehalogenase from Methylobacterium sp. CPA1.

DL-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (DL-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of DL-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6(3), with unit-cell parameters a = b = 186.2, c = 114.4 A. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V(M) value of 4.20-2.10 A3 Da(-1). A self-rotation function revealed peaks on the chi = 180 degrees section. X-ray data have been collected to 1.75 A resolution.