Engineering of Conserved Sequence Motif 1 Residues in Halohydrin Dehalogenase HheC Simultaneously Enhances Activity, Stability, and Enantioselectivity

Abstract

Halohydrin dehalogenases (HHDHs) are powerful enzymes for the asymmetric diversification of oxyfunctionalized synthons. They feature two characteristic sequence motifs that distinguish them from homologous short-chain dehydrogenases and reductases. Sequence motif 1, carrying a conserved threonine, glycine, and a central aromatic residue, lines the nucleophile binding pocket of HHDHs. It could therefore impact nucleophile binding and presumably also the activity of the enzymes. However, experimental evidence supporting this theory is largely missing. Herein, we systematically studied the mutability of the three conserved motif 1 residues as well as their resulting impact on enzyme activity, stability, and selectivity in two model HHDHs: HheC from Agrobacterium radiobacter AD1 and HheG from Ilumatobacter coccineus. In both HheC and HheG, the conserved threonine and glycine tolerated mutations to only structurally similar amino acids. In contrast, the central aromatic (i.e., phenylalanine or tyrosine) residue of motif 1 demonstrated much higher variability in HheC. Remarkably, some of these variants featured drastically altered activity, stability, and selectivity characteristics. For instance, variant HheC F12Y displayed up to 5-fold increased specific activity in various epoxide ring opening and dehalogenation reactions as well as enhanced enantioselectivity (e.g., an E-value of 74 in the azidolysis of epichlorohydrin compared to E = 22 for HheC wild type) while also exhibiting a 10 K higher apparent melting temperature. QM and MD simulations support the experimentally observed activity increase of HheC F12Y and reveal alterations in the hydrogen bonding network within the active site. As such, our results demonstrate that multiple enzyme properties of HHDHs can be altered through the targeted mutagenesis of conserved motif 1 residues. In addition, this work illustrates that motif 1 plays vital roles beyond nucleophile binding by impacting the solubility and stability properties. These insights advance our understanding of HHDH active sites and will facilitate their future engineering.