The Identification of Catalytic Pentad in the Haloalkane Dehalogenase DhmA from Mycobacterium avium N85: Reaction Mechanism and Molecular Evolution

Warning

This publication doesn't include Faculty of Economics and Administration. It includes Faculty of Science. Official publication website can be found on muni.cz.
Authors

PAVLOVÁ Martina KLVAŇA Martin JESENSKÁ Andrea PROKOP Zbyněk KONEČNÁ Hana SATO T. TSUDA M. NAGATA Yuji DAMBORSKÝ Jiří

Year of publication 2006
Type Article in Periodical
Magazine / Source JOURNAL OF STRUCTURAL BIOLOGY
MU Faculty or unit

Faculty of Science

Citation
Web http://loschmidt.chemi.muni.cz/peg/abstracts/jsb06.html
Field Biochemistry
Keywords Haloalkane dehalogenase DhmA; Mycobacterium avium N85; expression ; Site-directed mutagenesis
Description 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.
Related projects:

You are running an old browser version. We recommend updating your browser to its latest version.