title: In vivo screening of haloalkane dehalogenase mutants.
abstract: Haloalkane dehalogenase (Dh1A) from Xanthobacter autotrophicus GJ10 catalyzes the dehalogenation of short chain primary alkyl halides. Due to the high Km and low turnover, wild type Dh1A is not optimal for applications in bioremediation. We have developed an in vivo screen, based on a colorimetric pH indicator, to identify Dh1A mutant with improved catalytic activity. After screening 50,000 colonies, we identified a Dh1A mutant with a lower pH optimum. Sequence analysis of the mutant revealed a single substitution, alanine 149 to threonine, which is located close to the active site of Dh1A. Replacement of alanine 149 via site-directed mutagenesis with threonine, serine or cysteine retained the mutant phenotype. Other substitutions at position 149 show little or no activity.
authors.affiliation: Structural Biology Group, Los Alamos National Laboratory, NM 87545, USA.
authors.forename: C H
authors.initials: CH
authors.lastname: Chang
authors.forename: J F
authors.initials: JF
authors.lastname: Schindler
authors.forename: C J
authors.initials: CJ
authors.lastname: Unkefer
authors.forename: L A
authors.initials: LA
authors.lastname: Vanderberg
authors.forename: J R
authors.initials: JR
authors.lastname: Brainard
authors.forename: T C
authors.initials: TC
authors.lastname: Terwilliger
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005025:Ethylene Dichlorides / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D005025:Ethylene Dichlorides; D011994:Recombinant Proteins; C024565:ethylene dichloride; D006867:Hydrolases; C046137:haloalkane dehalogenase