title: 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 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.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Jitsumori
authors.forename: Rie
authors.initials: R
authors.lastname: Omi
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kurihara
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kurata
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Mihara
authors.forename: Ikuko
authors.initials: I
authors.lastname: Miyahara
authors.forename: Ken
authors.initials: K
authors.lastname: Hirotsu
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
mesh_terms: D000085:Acetates / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019117:Burkholderia / Q000737:chemistry* / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D005463:Fluoroacetates / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D000085:Acetates; D001426:Bacterial Proteins; D005463:Fluoroacetates; C006972:chloroacetic acid; D006867:Hydrolases; C023098:haloacetate dehalogenase