Halohydrin dehalogenases (HHDHs) have recently distinguished themselves as powerful enzymes for the asymmetric diversification of oxyfunctionalized synthons. In nature, these bacterial lyases catalyze the reversible dehalogenation of β-haloalcohols through formation of the corresponding epoxides. More importantly, they are capable of accepting a number of anionic C-, N-, O-, and S-nucleophiles in the reverse reaction, i.e. epoxide ring opening, giving access to a large repertoire of valuable products. For instance, recent impressive biocatalytic examples for the application of HHDHs in asymmetric synthesis include the preparation of enantiopure βnitroalcohols , and thiiranes , as well as the desymmetrization of 2-substituted-1,3-dichloro-2-propanols with subsequent cyanate-mediated ring opening to afford optically pure epoxides and oxazolidinones among others. 7,13,14 HHDHs share significant homology/similarity with short-chain dehydrogenases and reductases (SDRs) on the sequence, structural and mechanistic level as the result of a close phylogenetic relationship, although they catalyze entirely different chemical reactions. Previously, this similarity has largely impeded a fast discrimination of HHDHs and SDR enzymes solely based on given protein sequences. With the discovery of HHDH-specific sequence motifs in 2014, database mining approaches facilitated the identification of novel HHDHs and yielded a plethora of new enzymes with partially unprecedented catalytic characteristics. These HHDH sequence fingerprints include the N-terminal motif T-X4-F/Y-X-G (motif 1), lining the nucleophile binding pocket of halohydrin dehalogenases, as well as the motif S-X12-Y-X3-R (motif 2), covering the catalytic residues serine, tyrosine and arginine. For comparison, the corresponding sequence motifs of SDRs are T-G/A-X3-G/A-X-G (motif 1) and S-X10-14-Y-X3-K (motif 2), respectively. In contrast to HHDHs, SDR motif 1 represents the typical glycinerich nucleotide-binding sequence required for nicotinamide cofactor binding in those enzymes. While the mechanistic role of the three conserved catalytic residues serine, tyrosine and arginine (as part of motif 2) of HHDHs has already been elucidated using HheC from Agrobacterium radiobacter AD1 as a model enzyme, the functional role and mutability of the conserved residues in motif 1 remains largely unexplored. Moreover, we hypothesized that HHDH variants with substantially improved biocatalytic performance could be accessed through engineering of motif 1 residues, as they should impact nucleophile binding. Crystal structures of HheC in complex with a bromide or chloride ion (PDB IDs 1PWX and 1PWZ, respectively) have demonstrated that the side chain of the central aromatic residue F12 in motif 1 forms a direct interaction with the negatively charged halide, which is tightly wedged between aromatic residues. Such a direct interaction with the nucleophile, however, was not present in the crystal structure of HheB from Corynebacterium sp. strain N-1074 in complex with chloride (PDB ID 4ZD6), which carries a tyrosine at position 19. Moreover, in a study by Wu et al. on the thermostabilization of HheC by combinatorial directed evolution, mutation F12Y was found to increase the enzyme's thermostability and yielded a 1.5-fold gain in specific activity in the dehalogenation of 1,3-dichloropropanol. Similarly, mutations at the structurally equivalent position Y15 in AbHheG from Acidimicrobiia bacterium yielded variants with improved enantioselectivity in the ring opening of styrene oxide with cyanate quite recently. Both examples directly hint at the hitherto underexplored possibility to steer the catalytic properties of HHDHs via targeted mutagenesis of sequence motif 1.