Haloalkane dehalogenases catalyse the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilising residues that are important in substrate binding and stabilisation of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenase, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 possesses a unique halide-stabilising 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 enzyme's structure and catalytic properties 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, and of the stabilisation of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding 3.7-fold, presumably due to the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the enzyme's substrate specificity and reduced its K0.5 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 stabilises its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family. © 2013 The Authors Journal compilation © 2013 FEBS.