title: The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution.
abstract: Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.
authors.affiliation: Loschmidt Laboratories, Masaryk University, Brno, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Pavlová
authors.forename: Martin
authors.initials: M
authors.lastname: Klvana
authors.forename: Andrea
authors.initials: A
authors.lastname: Jesenská
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Prokop
authors.forename: Hana
authors.initials: H
authors.lastname: Konecná
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Jirí
authors.initials: J
authors.lastname: Damborský
mesh_terms: D020134:Catalytic Domain; D019143:Evolution, Molecular; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification; D009162:Mycobacterium avium / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
chemical_list: D050505:Mutant Proteins; D006867:Hydrolases; C046137:haloalkane dehalogenase