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The effect of a unique halide‐stabilizing residue on the catalytic properties of haloalkane dehalogenase D at A from A grobacterium tumefaciens C 58
Author(s) -
Hasan Khomaini,
Gora Artur,
Brezovsky Jan,
Chaloupkova Radka,
Moskalikova Hana,
Fortova Andrea,
Nagata Yuji,
Damborsky Jiri,
Prokop Zbynek
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12238
Subject(s) - chemistry , stereochemistry , active site , tryptophan , dehalogenase , hydrogen bond , catalysis , enzyme , biochemistry , molecule , organic chemistry , amino acid
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 D at A from Agrobacterium tumefaciens C 58 ( EC 3.8.1.5 ) possesses a unique halide‐stabilizing tyrosine residue, Y 109, in place of the conventional tryptophan. A variant of D at A with the Y 109 W 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 D at A 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.