Self-assembly enables formation of incredibly diverse supramolecular structures with practically important functions from simple and inexpensive building blocks. Here, we show how a semirational, bottom-up approach to create emerging properties can be extended to a design of highly enantioselective catalytic nanoassemblies. The designed peptides comprising as few as two amino acid residues spontaneously self-assemble in the presence of metal ions to form supramolecular, vesicle-like nanoassemblies that promote transfer hydrogenation of ketones in an aqueous phase with excellent conversion rates and enantioselectivities (>90% ee).

Directed evolution can rapidly achieve dramatic improvements in the properties of a protein or bestow entirely new functions on it. We have discovered a strong correlation between the probability of nding a productive mutation at a particular position of a protein and a chemical shift perturbation in Nuclear Magnetic Resonance spectra upon addition of an inhibitor for the chemical reaction it promotes. In a proof-of-concept study we converted myoglobin, a non-enzymatic protein, into the most active Kemp eliminase reported to date using only three mutations. The observed levels of catalytic e ciency are on par with the levels shown by natural enzymes. This simple approach, that requires no a priori structural or bioinformatic knowledge, is widely applicable and will unleash the full potential of directed evolution. Full TextDirected evolution is a powerful tool for improving existing properties and imparting completely new functionalities onto proteins. [1][2][3][4] Nonetheless, even in small proteins its potential is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20 100 combinations, which is currently beyond any existing experimental approach. Fortunately, in practice, selective modi cation of relatively few residues is su cient for e cient improvement, functional enhancement and repurposing of existing proteins. 5 Moreover, computational methods have been developed to predict the location, and, in certain cases, identities of potentially productive mutations. [6][7][8][9] Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer limited ability to identify bene cial mutations far from the active site, even though such changes may dramatically improve the catalytic properties of an enzyme. 10 Here we show that mutagenic hot spots in enzymes can be identi ed using Nuclear Magnetic Resonance (NMR) spectroscopy. In a proof-of-concept study we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly e cient Kemp eliminase using only three mutations. The observed levels of catalytic e ciency (k cat /K M of 2.8 x 10 6 M -1 s -1 and k cat /k uncat > 10 8 ) are the highest reported for any designed protein and are on par with the levels shown by natural enzymes for the reactions they are evolved to catalyze. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to unleash the full potential of directed enzyme evolution.Recent paradigm shifting advances in understanding the fundamental principles that drive enzyme evolution point to a major role of global conformational selection for productive arrangements of functional groups to perfect transition state stabilization, as well as steric and electrostatic interactio...

Minimalist enzymes designed to catalyze model reactions provide useful starting points for creating catalysts for practically important chemical transformations. We have shown that Kemp eliminases of the AlleyCat family facilitate conversion of leflunomide (an immunosuppressor pro‐drug) to its active form teriflunomide with outstanding rate enhancement (nearly four orders of magnitude) and catalytic proficiency (more than seven orders of magnitude) without any additional optimization. This remarkable activity is achieved by properly positioning the substrate in close proximity to the catalytic glutamate with very high pKa.

Directed evolution can rapidly achieve dramatic improvements in the properties of a protein or bestow entirely new functions on it. We have discovered a strong correlation between the probability of finding a productive mutation at a particular position of a protein and a chemical shift perturbation in Nuclear Magnetic Resonance spectra upon addition of an inhibitor for the chemical reaction it promotes. In a proof-of-concept study we converted myoglobin, a non-enzymatic protein, into the most active Kemp eliminase reported to date using only three mutations. The observed levels of catalytic efficiency are on par with the levels shown by natural enzymes. This simple approach, that requires no a priori structural or bioinformatic knowledge, is widely applicable and will unleash the full potential of directed evolution.

The Front Cover picture shows that AlleyCat2, a member of the AlleyCat family of allosterically regulated Kemp eliminases, is capable of binding leflunomide, an immunosuppressant drug, and converting it into teriflunomide, its active form, with remarkable efficiency. In their Communication, E. A. Caselle, J. H. Yoon et al. show that small libraries of designed catalysts provide fertile ground for discovering new reactivities. AlleyCat2 relies on a high pKa of the active base and proper positioning of the substrate in the hydrophobic cleft of the enzyme to promote catalysis. This work also demonstrates that using pH rate profiles to determine the pKa of the active residue can be quite misleading and NMR studies that can probe specific atoms directly provide invaluable mechanistic information. More information can be found in the Communication by E. A. Caselle, J. H. Yoon et al. on page 1425 in Issue 5, 2019 (DOI: 10.1002/cctc.201801994).

The front cover artwork for Issue 05/2019 is provided by the Korendoych and the Makhlynets Labs at Syracuse University (USA) in collaboration with the VIB Centre for Structural Biology (Belgium), College of Charleston (USA) and Kyiv National University (Ukraine). The image shows AlleyCat2, a minimalist allosterically regulated Kemp eliminase, efficiently promoting ring opening of leflunomide, an immunosuppresant. Small libraries of designed catalysts provide fertile ground for discovering new reactivities. See the Communication itself at https://doi.org/10.1002/cctc.201801994.

Directed evolution has emerged as a powerful tool for improving protein properties and imparting completely new functionalities onto existing proteins. Several computational methods have been successful in predicting potential hotspots for directed evolution. However, they rely heavily on prior structural and/or functional information. To address these fundamental limitations, we focused on Nuclear Magnetic Resonance (NMR) as a tool to rapidly evolve enzymes without structural characterization. AlleyCat, a computationally designed Kemp eliminase produced by introducing a single mutation on C‐terminal domain of a non‐enzymatic metalloprotein Calmodulin, has recently been evolved into AlleyCat7. This traditional directed evolution process required seven rounds of mutagenesis and screening. A retrospective analysis of the evolved variants of AlleyCat series showed a remarkable correlation between chemical shift perturbation and probability of finding a productive mutation in the vicinity of that residue. Using our novel approach, AlleyCat7 has been rapidly evolved into AlleyCat10 with an improvement of about four‐fold in catalytic efficiency after three successive rounds of evolution. NMR‐guided evolution strategy eliminates excessive mutagenesis and has the potential to unlock exciting avenues in directed evolution of protein catalysts.