title: Site-directed mutagenesis of the 2-haloalkanoic acid dehalogenase I gene from Pseudomonas sp. strain CBS3 and its effect on catalytic activity.
abstract: Two 2-haloalkanoic acid dehalogenases from Pseudomonas sp. strain CBS3 catalyse hydrolytic dehalogenation of chloroacetate and 2-chloropropionate. We used site-directed mutagenesis to introduce specific changes in the dehalogenase I encoding gene (dehCI). Substitution of Asp-10 by Ala-10 resulted in complete loss of dehalogenating activity although expression of the 2-haloacid dehalogenase I was not affected in the mutant as shown by western blot analysis, and although comparison of the mutated enzyme with the wild type enzyme indicated that extensive rearrangements in the three-dimensional structure of the enzyme had not occurred. From these data we suggest that Asp-10 of 2-haloacid dehalogenases I from Pseudomonas sp. strain CBS3 may be the nucleophilic residue in the active-site of this enzyme essential for halide release.
authors.affiliation: Institut für Mikrobiologie, Universität Hohenheim, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Schneider
authors.forename: R
authors.initials: R
authors.lastname: Müller
authors.forename: R
authors.initials: R
authors.lastname: Frank
authors.forename: F
authors.initials: F
authors.lastname: Lingens
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D007120:Immunochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D006867:Hydrolases; C034507:2-haloacid dehalogenase