Haloalkane dehalogenase from compost bacterium Saccharomonospora viridis DSM 43017 with unusual catalytic residues, unique S-enantiopreference and high thermostability.

Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.g., DsaA, DmxA, or DmrB remain catalytically active while employing a single halide-stabilizing residue. Here we describe a novel haloalkane dehalogenase DsvA from a mild thermophilic bacterium Saccharomonospora viridis DSM 43017 possessing one canonical halide-stabilizing tryptophan (W125). At the position of the second halide-stabilizing residue, it contains the phenylalanine (F165), which can not stabilize the halogen anion released during the enzymatic reaction by a hydrogen-bond. Based on the sequence and structural alignments, we identified a putative second halide-stabilizing tryptophan (W162) located on the same α-helix as F165, but on the opposite side of the active site. The potential involvement of this residue in DsvA catalysis was investigated by the construction and biochemical characterization of the three variants, DsvA01 (F165W), DsvA02 (W162F) and DsvA03 (W162F+F165W). Interestingly, DsvA exhibits a preference for the (S)- over the (R)-enantiomers of β-bromoalkanes, which has not been reported before for any characterized haloalkane dehalogenase. Moreover, DsvA shows remarkable operational stability at elevated temperatures. The present study illustrates that protein sequences possessing the unconventional composition of catalytic residues represent a valuable source of novel biocatalysts. IMPORTANCE The present study describes a novel haloalkane dehalogenase DsvA originated from a mild thermophilic bacterium Saccharomonospora viridis DSM 43017. We report its high thermostability, remarkable operational stability at high temperatures, and a (S)-enantiopreference, which makes this enzyme an attractive biocatalyst for practical applications. A sequence analysis uncovered that DsvA possesses an unusual composition of halide-stabilizing tryptophan residues in its active site. We constructed and biochemically characterized two single-point and one double-point mutant and identified the non-canonical halide-stabilizing residue. Our study underlines the importance of searching also for non-canonical catalytic residues in protein sequences.