Artificial metalloenzymes (ArMs), which combine an abiotic metal cofactor with a protein scaffold, catalyze various synthetically useful transformations. To complement the natural enzymes' repertoire, effective optimization protocols to improve ArM's performance are required. Here we report on our efforts to optimize the activity of an artificial transfer hydrogenase (ATHase) using E. coli whole cells. For this purpose, we rely on a self-immolative quinolinium substrate which, upon reduction, releases fluorescent umbelliferone, thus allowing efficient screening. Introduction of a loop in the immediate proximity of the Ircofactor afforded an ArM with up to fivefold increase in transfer hydrogenation activity compared to the wild-type ATHase using purified mutants.

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.

No abstract

An artificial metathesase results from incorporation of an Hoveyda-Grubbs catalyst bearing an arylsulfonamide anchor within human carbonic anhydrase II. The optimization of the catalytic performance is achieved upon combining both chemical and genetic means. Up to 28 TONs were obtained within four hours under aerobic physiological conditions.

Co Cr Pt – Si O 2 perpendicular recording media containing dual-Ru intermediate layers have been studied. The bottom Ru layer deposited under a higher mobility condition, i.e., low Ar gas pressure and bias on substrate, helps to achieve a good crystalline texture, and the top Ru layer under a lower mobility condition, i.e., high Ar gas pressure, promotes the formation of a columnar microstructure. Three kinds of grain boundaries have been observed. The nucleation field and the coercivity as a function of the thickness and dc bias of the bottom Ru layer have been investigated. Medium noise reduction has been observed for dual-Ru intermediate layer media (dual-Ru media) in comparison with that of single intermediate layer media (single-Ru media). This is mainly attributed to the optimization of the physical grain segregation.