Chiral amines can be made by insertion of a carbene into an N-H bond using two-catalyst systems that combine a transition metal carbene-transfer catalyst and a chiral proton-transfer catalyst to enforce stereocontrol. Haem proteins can effect carbene N-H insertion, but asymmetric protonation in an active site replete with proton sources is challenging. Here we describe engineered cytochrome P450 enzymes that catalyze carbene N-H insertion to prepare biologically relevant α-amino lactones with high activity and enantioselectivity (up to 32,100 total turnovers, >99% yield and 98% e.e.). These enzymes serve as dual-function catalysts, inducing carbene transfer and promoting the subsequent proton transfer with excellent stereoselectivity in a single active site. Computational studies uncover the detailed mechanism of this new-to-nature enzymatic reaction and explain how active-site residues accelerate this transformation and provide stereocontrol.Amines are ubiquitous in bioactive molecules and functional materials 1,2 , and the development of efficient and selective methods for C-N bond construction remains one of the central themes of modern organic chemistry and biochemistry 3-5 . Among the numerous ways to construct C-N bonds, carbene insertion into N-H bonds 6-10 benefits from the high reactivity of carbene species and excellent functional group compatibility to rapidly build complex nitrogen-containing molecules. In the last several years, empowered by directed evolution, metallo-haem-dependent enzymes (cytochromes P450, cytochromes c and globins, for example) have exhibited an impressive ability to catalyze non-natural carbene-and nitrene-transfer reactions with high efficiency and selectivity. Specifically, haem proteins have been engineered to perform carbene N-H insertion reactions with catalytic efficiency far exceeding their small-molecule counterparts (up to thousands of total turnover numbers (TTN)) [11][12][13][14] . However, compared to cyclopropanation 15 , C-H insertion 16 and many other carbene transfer reactions also catalyzed by haem proteins 17,18 , N-H insertion reactions are still underdeveloped, especially with respect to high stereocontrol.In small-molecule catalysis, a common strategy for asymmetric N-H insertion is to employ a transition-metal catalyst for carbene transfer along with a separate chiral proton-transfer catalyst (PTC) for stereoinduction (Fig. 1a) 19,20 . The carbene precursor first reacts to form a metal carbene species, which can be trapped by the amine substrate through nucleophilic attack, generating an ylide intermediate. The asymmetric protonation of the ylide is then guided by a chiral PTC, such as a chiral phosphoric acid 19 or amino-thiourea 20 ; other proton sources need to be strictly avoided to ensure high asymmetric induction. Computational studies by Shaik and coworkers 21 have revealed a similar mechanism for haem protein-catalyzed N-H insertion reactions. Thus, the challenge in achieving high enantioselectivity originates from the difficulty in precisely contr...

The efficient regeneration of cofactors is vital for the establishment of biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO 2 ). However, formate dehydrogenases (FDHs) are usually specific to NAD + , such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP + , but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP + , specificity toward NADP + , and affinity toward formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits practically no activity toward NADP + , we generate a library of >10 6 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as the sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 14-fold higher specificity in comparison to the best enzyme previously engineered, while retaining high affinity toward formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how nonadditive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify highly proficient enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond time scale interconversion between these functional states hamper their experimental and computational characterization. Here, we combine extensive molecular dynamics simulations, enhanced sampling techniques, and dynamical networks to describe the allosteric activation of imidazole glycerol phosphate synthase (IGPS) from the substrate-free form to the active ternary complex. IGPS is a heterodimeric bienzyme complex whose HisH subunit is responsible for hydrolyzing glutamine and delivering ammonia for the cyclase activity in HisF. Despite significant advances in understanding the underlying allosteric mechanism, essential molecular details of the long-range millisecond allosteric activation of IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound in HisF, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that the combined effector and substrate binding dramatically decreases the conformational barrier associated with oxyanion hole formation, in line with the experimentally observed 4500-fold activity increase in glutamine hydrolysis. The allosteric activation is controlled by correlated time-evolving dynamic networks connecting the effector and substrate binding sites. This computational strategy tailored to describe millisecond events can be used to rationalize the effect of mutations on the allosteric regulation and guide IGPS engineering efforts.

With innumerable clinical failures of target-specific drug candidates for multifactorial diseases, such as Alzheimer’s disease (AD), which remains inefficiently treated, the advent of multitarget drug discovery has brought a new breath of hope. Here, we disclose a class of 6-chlorotacrine (huprine)–TPPU hybrids as dual inhibitors of the enzymes soluble epoxide hydrolase (sEH) and acetylcholinesterase (AChE), a multitarget profile to provide cumulative effects against neuroinflammation and memory impairment. Computational studies confirmed the gorge-wide occupancy of both enzymes, from the main site to a secondary site, including a so far non-described AChE cryptic pocket. The lead compound displayed in vitro dual nanomolar potencies, adequate brain permeability, aqueous solubility, human microsomal stability, lack of neurotoxicity, and it rescued memory, synaptic plasticity, and neuroinflammation in an AD mouse model, after low dose chronic oral administration.

The pharmacological inhibition of soluble epoxide hydrolase (sEH) is efficient for the treatment of inflammatory and pain-related diseases. Numerous potent sEH inhibitors (sEHIs) present adamantyl or phenyl moieties, such as the clinical candidates AR9281 or EC5026. Herein, in a new series of sEHIs, these hydrophobic moieties have been merged in a benzohomoadamantane scaffold. Most of the new sEHIs have excellent inhibitory activities against sEH. Molecular dynamics simulations suggested that the addition of an aromatic ring into the adamantane scaffold produced conformational rearrangements in the enzyme to stabilize the aromatic ring of the benzohomoadamantane core. A screening cascade permitted us to select a candidate for an in vivo efficacy study in a murine model of cerulein-induced acute pancreatitis. The administration of 22 improved the health status of the animals and reduced pancreatic damage, demonstrating that the benzohomoadamantane unit is a promising scaffold for the design of novel sEHIs.