X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from Burkholderia sp. Strain FA1

Abstract

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 ␣/␤ 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 1 atom of Trp150 and the N 2 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.Fluoroacetate is a naturally occurring organofluorine compound. An actinomycete, "Streptomyces cattleya" (30), and some plants in Australia and Africa (2) produce this highly toxic compound. Fluoroacetate has a carbon-fluorine bond, whose dissociation energy is among the highest found in nature (8). Despite this fact, fluoroacetate dehalogenases (FAcDEXs) from Burkholderia sp. strain FA1 (FAc-DEX FA1) (20) and Delftia acidovorans strain B (formerly Moraxella sp. strain B; FAc-DEX H1) (16) catalyze the hydrolytic defluorination of fluoroacetate to produce glycolate. These enzymes are unique in that they catalyze the cleavage of the carbon-fluorine bond, which is much stronger than other carbon-halogen bonds.FAc-DEXs show weak but significant sequence similarity to proteins that belong to the ␣/␤ hydrolase superfamily, such as epoxide hydrolases from Agrobacterium radiobacter AD1 (37) and humans and haloalkane dehalogenases from Sphingobium japonicum UT26 (formerly Sphingomonas paucimobilis UT26) (32) and Xanthobacter autotrophicus GJ10 (17). Although haloalkane dehalogenases of this superfamily catalyze the hydrolytic cleavage of carbon-halogen bonds, none of them catalyzes the cleavage of a carbon-fluorine bond. Dehalogenases that belong to other families, such as L-2-haloacid dehalogenase (22), DL-2-haloacid dehalogenase (28), and haloalcohol dehalogenase (42), also do not catalyze the cleavage of a carbon-...

Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

“…The dimerization interface encompasses an average surface area of 1,365 Å 2 on each monomer. Dimerization is a feature commonly seen in the crystal structures of bacterial ␣/␤ hydrolases (29,43,57).…”

“…While confirming the predicted similarity between ArEH and Cif, our distance matrix alignment also revealed that the most closely related known structure was actually that of the fluoroacetate dehalogenase from Burkholderia sp. strain FA1 (FAc-DEX FA1), with a Z score of 35.7 and an rmsd of 1.9 Å. FAc-DEX FA1 is an ␣/␤ hydrolase that defluorinates fluoroacetate to form glycolic acid (29), thus detoxifying this compound, which can form a potent aconitase inhibitor if ingested (24). As a family, the HADs are closely related in sequence to the EHs and also utilize an aspartic acid side chain for initial nucleophilic attack (52).…”

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

“…46) The following mutant enzymes showed no activity toward fluoroacetate and chloroacetate: F34A, D104A, R105A, R108A, D128A, Y147A, H149A, W179A, Y212A, H271A, and F272A. In contrast, replacement of Trp150 with Ala did not significantly affect the activity toward chloroacetate, whereas that toward fluoroacetate was completely lost due to this mutation.…”

“…40) Mutation of Asp104 and His271 of FAc-DEX FA1 resulted in a total loss of the enzyme activity, suggesting that the reaction mechanism is conserved between these enzymes. 46) To investigate the reaction mechanism of fluoroacetate dehalogenase further, the crystal structure of FAc-DEX FA1 was determined. 46) This enzyme belongs to the / hydrolase superfamily and shares three-dimensional structural similarity with epoxide hydrolase and haloalkane dehalogenase.…”

“…46) To investigate the reaction mechanism of fluoroacetate dehalogenase further, the crystal structure of FAc-DEX FA1 was determined. 46) This enzyme belongs to the / hydrolase superfamily and shares three-dimensional structural similarity with epoxide hydrolase and haloalkane dehalogenase. 28) FAc-DEX FA1 exists as a homodimer, and each subunit consists of core and cap domains.…”