Breaking Symmetry: Engineering Single-Chain Dimeric Streptavidin as Host for Artificial Metalloenzymes

Abstract

The biotin–streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17 000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.

Enabling protein-hosted organocatalytic transformations

“…14a). Hence, residues from both monomers can interact with the catalyst and substrates, 141 and the docking studies revealed that the S112Y mutation from each monomer, namely S112Y A and S112Y B , is essential to the catalysis by ligand 14.…”

Artificial imine reductases: developments and future directions

“…Most monovalent scdSav artIREDs were found to outperform their equivalent divalent scd artIREDs, among them, the best variants for producing (R)-3b (91% e.e., 100% conversion, 0.05 mol% catalyst loading), (R)-4b (96% e.e., 99% conversion, 0.05 mol% catalyst loading) and (S)-5b (91% e.e., 98% conversion, 0.5 mol% catalyst loading). 51 Directed evolution. Although other design strategies had initially had some success, it has proved challenging to predict the subtle effects of protein structure and bonding on enantioselectivity.…”

Exploring and Adapting the Molecular Selectivity of Artificial Metalloenzymes

“…For iridium-based ArMs, the majority of literature studies thus far have extensively focused on their use as artificial transfer hydrogenases (ATHase). [113][114][115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130][131][132] In these works, asymmetric transfer hydrogenation has been shown possible for a number of substrates that include imines, enones, ketones, and for NAD + reduction. One intriguing example of an ATHase that has been used with biological systems relies on a cell-surface display system that shuttles streptavidin into the E. coli periplasm ( Figure 5A).…”