title: Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B.
abstract: Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon-fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the alpha-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.
authors.affiliation: Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Ichiyama
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kurihara
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Kogure
authors.forename: Susumu
authors.initials: S
authors.lastname: Tsunasawa
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Kawasaki
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D005463:Fluoroacetates / Q000378:metabolism; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009016:Moraxella / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D020673:Protein Renaturation
chemical_list: D005463:Fluoroacetates; D001216:Asparagine; C014815:fluoroacetic acid; D006867:Hydrolases; C023098:haloacetate dehalogenase