Structural, Kinetic, and Docking Studies of Artificial Imine Reductases Based on Biotin–Streptavidin Technology: An Induced Lock-and-Key Hypothesis

An artificial imine reductase results upon incorporation of a biotinylated Cp*Ir moiety (Cp* = C5Me5 –) within homotetrameric streptavidin (Sav) (referred to as Cp*Ir­(Biot-p-L)­Cl] ⊂ Sav). Mutation of S112 reveals a marked effect of the Ir/streptavidin ratio on both the saturation kinetics as well as the enantioselectivity for the production of salsolidine. For [Cp*Ir­(Biot-p-L)­Cl] ⊂ S112A Sav, both the reaction rate and the selectivity (up to 96% ee (R)-salsolidine, k cat 14–4 min–1 vs [Ir], K M 65–370 mM) decrease upon fully saturating all biotin binding sites (the ee varying between 96% ee and 45% ee R). In contrast, for [Cp*Ir­(Biot-p-L)­Cl] ⊂ S112K Sav, both the rate and the selectivity remain nearly constant upon varying the Ir/streptavidin ratio [up to 78% ee (S)-salsolidine, k cat 2.6 min–1, K M 95 mM]. X-ray analysis complemented with docking studies highlight a marked preference of the S112A and S112K Sav mutants for the S Ir and R Ir enantiomeric forms of the cofactor, respectively. Combining both docking and saturation kinetic studies led to the formulation of an enantioselection mechanism relying on an “induced lock-and-key” hypothesis: the host protein dictates the configuration of the biotinylated Ir-cofactor which, in turn, by and large determines the enantioselectivity of the imine reductase.