The Identification of Catalytic Pentad in the Haloalkane Dehalogenase DhmA from Mycobacterium avium N85: Reaction Mechanism and Molecular Evolution
|Original title:||The Identification of Catalytic Pentad in the Haloalkane Dehalogenase DhmA from Mycobacterium avium N85: Reaction Mechanism and Molecular Evolution|
|Authors:||Martina Pavlová, Martin Klvaňa, Andrea Jesenská, Zbyněk Prokop, Hana Konečná, T. Sato, M. Tsuda, Yuji Nagata, Jiří Damborský|
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.