A Dual Anchoring Strategy for the Directed Evolution of Improved Artificial Transfer Hydrogenases Based on Carbonic Anhydrase

International audienc

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

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 >17âEuro¯000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications

Convergent Evolution of Fungal Cysteine Dioxygenases

Cupin-type cysteine dioxygenases (CDOs) are non-heme iron enzymes that occur in animals, plants, bacteria and in filamentous fungi. In this report, we show that agaricomycetes contain an entirely unrelated type of CDO that emerged by convergent evolution from enzymes involved in the biosynthesis of ergothioneine. The activity of this CDO type is dependent on the ergothioneine precursor N-α-trimethylhistidine. The metabolic link between ergothioneine production and cysteine oxidation suggests that the two processes might be part of the same chemical response in fungi, for example against oxidative stress

An Artificial [Fe₄S₄]-Containing Metalloenzyme for the Reduction of CO₂ to Hydrocarbons

Iron–sulfur clusters have been reported to catalyze various redox transformations, including the multielectron reduction of CO2 to hydrocarbons. Herein, we report the design and assembly of an artificial [Fe4S4]-containing Fischer–Tropschase relying on the biotin–streptavidin technology. For this purpose, we synthesized a bis-biotinylated [Fe4S4] cofactor with marked aqueous stability and incorporated it in streptavidin. The effect of the second coordination sphere provided by the protein environment was scrutinized by cyclic voltammetry, highlighting the accessibility of the doubly reduced [Fe4S4] cluster. The Fischer–Tropschase activity was improved by chemo-genetic means for the reduction of CO2 to hydrocarbons with up to 14 turnovers

Spiers Memorial Lecture: Shielding the active site: a streptavidin superoxide-dismutase chimera as a host protein for asymmetric transfer hydrogenation

By anchoring a metal cofactor within a host protein, so-called artificial metalloenzymes can be generated. Such hybrid catalysts combine the versatility of transition metals in catalyzing new-to-nature reactions with the power of genetic-engineering to evolve proteins. With the aim of gaining better control over second coordination-sphere interactions between a streptavidin host-protein (Sav) and a biotinylated cofactor, we engineered a hydrophobic dimerization domain, borrowed from superoxide dismutase C (SOD), on Sav's biotin-binding vestibule. The influence of the SOD dimerization domain (DD) on the performance of an asymmetric transfer hydrogenase (ATHase) resulting from anchoring a biotinylated Cp*Ir-cofactor - [Cp*Ir(biot- p -L)Cl] ( 1 -Cl) - within Sav-SOD is reported herein. We show that, depending on the nature of the residue at position Sav S112, the introduction of the SOD DD on the biotin-binding vestibule leads to an inversion of configuration of the reduction product, as well as a fivefold increase in catalytic efficiency. The findings are rationalized by QM/MM calculations, combined with X-ray crystallography

Artificial Metalloenzyme-Catalyzed Enantioselective Amidation via Nitrene Insertion in Unactivated C(sp³)–H Bonds

Enantioselective C–H amidation offers attractive means to assemble C–N bonds to synthesize high-added value, nitrogen-containing molecules. In recent decades, complementary enzymatic and homogeneous-catalytic strategies for C–H amidation have been reported. Herein, we report on an artificial metalloenzyme (ArM) resulting from anchoring a biotinylated Ir-complex within streptavidin (Sav). The resulting ArM catalyzes the enantioselective amidation of unactivated C(sp3)–H bonds. Chemogenetic optimization of the Ir cofactor and Sav led to significant improvement in both the activity and enantioselectivity. Up to >700 TON and 92% ee for the amidation of unactivated C(sp3)–H bonds was achieved. The single crystal X-ray analysis of the artificial nitrene insertase (ANIase) combined with quantum mechanics-molecular mechanics (QM-MM) calculations sheds light on critical second coordination sphere contacts leading to improved catalytic performance

An Artificial Peroxidase based on the Biotin-Streptavidin Technology that Rivals the Efficiency of Natural Peroxidases

Horseradish peroxidase (HRP) is an archetypal heme-containing metalloenzyme that uses peroxide to oxidize various substrates. Capitalizing on a well-established catalytic mechanism, diverse peroxidase mimics have been widely investigated and optimized. Herein, we report on the design, assembly, characterization, and genetic engineering of an artificial heme-based peroxidase relying on the biotin-streptavidin technology. The crystal structure of both the wild-type and the best-performing double mutant of artificial peroxidase provided valuable insight regarding the nearby residues that were strategically mutated to optimize the peroxidase activity (i.e. Sav S112E K121H). We hypothesize that these two residues mimic the two key second coordination residues involved in activating the bound peroxide in HRP (i.e. Arg 38 and His 42). The evolved artificial peroxidase exhibited best-in-class activity for oxidizing two standard substrates (TMB and ABTS) in the presence of hydrogen peroxide

Artificial Metalloenzyme-Catalyzed Enantioselective Amidation via Nitrene Insertion in Unactivated C(<i>sp</i>³)–H Bonds

Enantioselective C–H amidation offers attractive means to assemble C–N bonds to synthesize high-added value, nitrogen-containing molecules. In recent decades, complementary enzymatic and homogeneous-catalytic strategies for C–H amidation have been reported. Herein, we report on an artificial metalloenzyme (ArM) resulting from anchoring a biotinylated Ir-complex within streptavidin (Sav). The resulting ArM catalyzes the enantioselective amidation of unactivated C(sp3)–H bonds. Chemogenetic optimization of the Ir cofactor and Sav led to significant improvement in both the activity and enantioselectivity. Up to >700 TON and 92% ee for the amidation of unactivated C(sp3)–H bonds was achieved. The single crystal X-ray analysis of the artificial nitrene insertase (ANIase) combined with quantum mechanics-molecular mechanics (QM-MM) calculations sheds light on critical second coordination sphere contacts leading to improved catalytic performance