Expanding the Substrate Scope of Native Amine Dehydrogenases through <i>In Silico</i> Structural Exploration and Targeted Protein Engineering
Laurine Ducrot, Megan Bennett, Gwenaëlle André, Eddy Elisée, Sacha Marynberg, Aurélie Fossey‐Jouenne, Anne Zaparucha, Gideon Grogan, Carine Vergne‐Vaxelaire
Abstract
Abstract Native Amine Dehydrogenases (nat‐AmDHs) are NAD(P)H‐enzymes performing reductive amination, mainly active towards small aliphatic aldehydes and cyclic ketones, due to active site volumes limited by the presence of several bulky amino acids. Herein, inspired by the diversity of residues at these positions among the family, we report the implementation of mutations F140A and W145A in Cfus AmDH and their transposition in nine other members. Moderate to high conversions were obtained with substrates not accepted by the native enzymes, notably n ‐alkylaldehydes (44.6 %–99.5 % for hexanal to nonanal) and n ‐alkylketones (16.0 %–53.7 % for hexan‐2‐one to nonan‐2‐one) with retention of excellent ( S )‐enantioselectivity (>99 % ee) . Complementary to the reported ( R )‐selective AmDHs, the promising mutant Cfus AmDH−W145A was further characterized for its synthetic potential. Crystal structure resolution and molecular dynamics gave insights into the cofactor and substrate specificity and the whole structural dynamics, thus providing keys for mutagenesis work on this enzyme family.