title: The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58.
abstract: Haloalkane dehalogenases catalyze the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K(0.5) for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.
authors.affiliation: Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Brno, Czech Republic.
authors.forename: Khomaini
authors.initials: K
authors.lastname: Hasan
authors.forename: Artur
authors.initials: A
authors.lastname: Gora
authors.forename: Jan
authors.initials: J
authors.lastname: Brezovsky
authors.forename: Radka
authors.initials: R
authors.lastname: Chaloupkova
authors.forename: Hana
authors.initials: H
authors.lastname: Moskalikova
authors.forename: Andrea
authors.initials: A
authors.lastname: Fortova
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Jiri
authors.initials: J
authors.lastname: Damborsky
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Prokop
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D004795:Enzyme Stability; D006219:Halogens / Q000737:chemistry / Q000378:metabolism*; D006846:Hydrocarbons, Halogenated / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D025341:Principal Component Analysis; D011487:Protein Conformation; D011789:Quantum Theory; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D006219:Halogens; D006846:Hydrocarbons, Halogenated; D050505:Mutant Proteins; D014443:Tyrosine; D006867:Hydrolases; C046137:haloalkane dehalogenase