R.B. Leveson-Gower

Hydrogen atom transfer (HAT) constitutes a powerful mechanism exploited in biology and chemistry alike to functionalize otherwise inert C(sp3)–H bonds in organic molecules. Despite its synthetic potential, achieving stereocontrol in chemical HAT-mediated C–H functionalization transformations remains challenging. By merging the radical reactivity of thiamine (ThDP)-dependent enzymes with chemical hydrogen atom transfer, we report here a photobiocatalytic strategy for the enantioselective C(sp3)–H acylation of an organic substrate, a transformation not found in nature, nor currently attainable by chemical means. This method enables the direct functionalization of benzylic C(sp3)–H sites in a broad range of substrates to furnish valuable enantioenriched ketone motifs with good to high enantioselectivity (up to 96% e.e.). Mechanistic and reactivity studies support the involvement of radical species derived from the Breslow intermediate and C–H substrate, along with the critical role of the photocatalyst and hydrogen atom abstraction reagent for productive catalysis. This study illustrates the productive integration of ThDP-mediated biocatalysis with chemical HAT, expanding the range of asymmetric C(sp3)–H functionalization transformations accessible through biocatalysis.

Sequential incorporation of an organic photocatalytic cofactor and a metal cofactor into streptavidin leads to artificial metalloenzymes (ArMs) that catalyze tandem abiotic transformations such as enantioselective formal C−H hydroxylation and photooxidation-Michael addition. This work introduces a programmable approach for the construction of ArMs that can catalyze tandem abiotic reactions.

Abstract

Artificial metalloenzymes (ArMs) enable the integration of abiotic cofactors within a native protein scaffold, allowing for non-natural catalytic activities. Previous ArMs, however, have primarily relied on single cofactor systems, limiting them to only one catalytic function. Here we present an approach to construct ArMs embedding two catalytic cofactors based on the biotin-streptavidin technology. By incorporating multiple catalytic cofactors into the four binding sites of streptavidin, we engineered programmable ArMs for tandem abiotic transformations including an enantioselective formal C−H hydroxylation and a photooxidation-Michael addition. This work thus outlines a promising strategy for the development of ArMs embedding multiple cofactors.

Nature, Published online: 06 January 2025; doi:10.1038/d41586-025-00011-8

A trove of data is providing insights into the main reasons studies are pulled from the arXiv preprint platform.

Proceedings of the National Academy of Sciences, Volume 122, Issue 1, January 2025.
SignificanceSingle-component flavin-dependent halogenases (FDHs) are attractive biocatalysts for halogenation. However, their underlying mechanisms of flavin chemistry remained unexplored. This work reports pre-steady-state kinetics of a single-component ...

Amide bond synthetase (ABS) enzymes catalyse amide formation in an environmentally friendly manner. This study advances the application of ABS by integrating these enzymes with other compatible biocatalysts, creating enantioselective and scalable cascade reactions to produce valuable amide products from abundant nitrile precursors. Furthermore, two innovative methods for C−H bond amidation of aromatic compounds are introduced.

Abstract

Amide bond formation is fundamental in nature and is widely used in the synthesis of pharmaceuticals and other valuable products. Current methods for amide synthesis are often step and atom inefficient, requiring the use of protecting groups, deleterious reagents and organic solvents that create significant waste. The development of cleaner and more efficient catalytic methods for amide synthesis remains an urgent unmet need. Herein, we present novel biocatalytic cascade reactions for synthesising various amides under mild aqueous conditions from readily available organic nitriles combining nitrile hydrolysing enzymes and amide bond synthetase enzymes. These cooperative biocatalytic cascades enable kinetic resolution of racemic nitriles and provide a highly enantioselective biocatalytic extension of the Strecker reaction. The regioselective non-directed C−H bond amidation of simple arenes was demonstrated through the incorporation of photoredox catalysis to the front end of the cascade. C−H bond amidation of simple aromatic precursors was also achieved via a CO₂ fixation cascade combining enzymatic carboxylation and amide bond synthesis in one-pot.

Nature, Published online: 15 January 2025; doi:10.1038/s41586-025-08611-0

Engineered enzymes for enantioselective nucleophilic aromatic substitutions

Herein we describe a set of privileged and stereocomplementary ene-reductase enzymes which, when induced by light and aided by an exogenous photocatalyst, catalyze the coupling of (hetero)aryl halides and alkenes in an asymmetric intermolecular hydroarylation process. Thus, carbon scaffolds containing C(sp2)-C(sp3) bonds are synthesized enzymatically from simple precursors in excellent enantiomeric excess. Furthermore, an intramolecular coupling is achieved through tethering of (hetero)aryl halides to their alkene reaction partners and in this manner problematic side reactions are suppressed and yields improved. This work extends the utility of photo-induced biocatalysis through the addition of the novel and pharmaceutically important (hetero)aromatic halide class of radical precursors.

A new-to-nature enzymatic platform for the enantioselective trifluoromethylazidation of alkenes has been successfully established. Through 11 rounds of directed evolution, an engineered variant of nonheme iron enzyme, BsQueD-CF_3, was developed, enabling the production of a wide range of enantioenriched CF₃-containing molecules. This platform based on metalloenzymes would open a new avenue for biocatalytic trifluoromethylation chemistry.

Abstract

Organofluorines, particularly those containing trifluoromethyl (CF₃) groups, play a critical role in medicinal chemistry. While trifluoromethylation of alkenes provides a powerful synthetic route to construct CF₃-containing compounds with broad structural and functional diversity, achieving enantioselective control in these reactions remains a formidable challenge. In this study, we engineered a nonheme iron enzyme, quercetin 2,3-dioxygenase from Bacillus subtilis (BsQueD), for the enantioselective trifluoromethylazidation of alkenes. Through directed evolution, the final variant BsQueD-CF₃ exhibited excellent enantioselectivity, with an enantiomeric ratio (e.r.) of up to 98 : 2. Preliminary mechanistic studies suggest the involvement of radical intermediates. This work expands biocatalytic organofluorine chemistry by reprogramming metalloenzymes for innovative trifluoromethylation reactions.

Nature, Published online: 22 January 2025; doi:10.1038/s41586-024-08455-0

A massively parallel assay developed to map the essential photosynthetic enzyme rubisco showed that non-trivial biochemical changes and improvements in CO2 affinity are possible, signposting further enzyme engineering efforts to increase crop yields.

Amino-acid residues distant from an enzymes active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the structural, functional, and mechanistic impacts of distal and active-site mutations discovered through directed evolution of the computationally designed retro-aldolase RA95. Active-site mutations improve catalytic efficiency by 3,600-fold, while distal mutations alone offer no improvement. When combined with active-site mutations, distal mutations further increase efficiency by 6-fold, demonstrating an epistatic effect. X-ray crystallography and molecular dynamics simulations reveal that distal mutations promote active site opening by altering loop dynamics. Kinetic solvent viscosity effects and electrostatic analysis show that distal mutations accelerate the chemical transformation by 100-fold, shifting the rate-limiting step to product release, which is further accelerated by the increased opening of the active site. These findings highlight the critical role of distal residues in shaping the active-site environment and facilitating the structural dynamics essential for progression through the catalytic cycle.

Promiscuity guided evolution of the decarboxylative aldolase, UstD, resulted in an efficient biocatalyst for synthesis of tertiary γ-hydroxy non-canonical amino acids. Simultaneous collection of variant activity and promiscuity was enabled by competition screening during directed evolution. Changes in promiscuity effectively identified distal residues that influence catalysis, a longstanding challenge in protein engineering.

Abstract

Many applications of enzymes benefit from activity on structurally diverse substrates. Here, we sought to engineer the decarboxylative aldolase UstD to perform a challenging C−C bond forming reaction with ketone electrophiles. The parent enzyme had only low levels of activity, portending multiple rounds of directed evolution and a possibility that mutations may inadvertently increase the specificity of the enzyme for a single model screening substrate. We show how to intentionally guide UstD towards generality through multi-generational directed evolution using substrate-multiplexed screening (SUMS). Mutations outside of the active site that impact catalytic function were immediately revealed by shifts in promiscuity, even when the overall activity was lower. By re-targeting these distal residues that couple to the active site with saturation mutagenesis, broadly activating mutations were readily identified. When analyzing active site mutants, SUMS identified both specialist enzymes that would have more limited utility as well as generalist enzymes with complementary activity on diverse substrates. These new UstD enzymes catalyze convergent synthesis of non-canonical amino acids bearing tertiary alcohol side chains. This methodology is easy to implement and enables the rapid and effective evolution of enzymes to catalyze desirable new functions.

Biocatalysis contributes significantly to the development of more sustainable synthetic pathways by using mild reaction conditions and water as a solvent. However, many relevant classes of compounds, including privileged groups in drug design, are not yet accessible via enzymatic pathways. In this context, the development of an enzymatic route to indazoles remains an unmet challenge. Here, we present the first example of nitroreductase-triggered indazole formation, in which 2-nitrobenzylamine derivatives are converted to reactive nitrosobenzylamine intermediates that spontaneously cyclize and aromatize to indazoles. Two nitroreductases, NfsA and BaNTR1, were found to accept a series of 2-nitrobenzylamine derivatives with excellent conversions (up to >99 %). In the case of N-substituted nitrosobenzylamines, 2H-indazoles were formed, whereas other derivatives led to 1H-indazoles. The synthetic value of the nitroreductase-triggered indazole formation was further demonstrated by successful coupling with an imine reductase (IRED15) in a sequential cascade reaction. With this cascade, N-methyl-2Hindazole was accessible from cheap 2-nitrobenzaldehyde and methylamine, resulting in 62 % isolated yield.

Nature, Published online: 12 February 2025; doi:10.1038/s41586-024-08553-z

A metalloenzyme capable of oxidatively cleaving cellulose, found in a microbial community specialized in lignocellulose degradation, could enable sustainable biofuel production.

An efficient artificial copper-dependent Michaelase featuring a metal-binding unnatural amino acid, e. g. bipyridyl alanine (BpyA), was optimized through directed evolution and applied in catalytic asymmetric additions of 2-acetyl azaarenes to nitroalkenes. Various chiral γ-nitro butyric acid derivatives were obtained in good yields with high enantioselectivities. Moreover, the reaction was performed at preparative scale and the product was used in follow-up derivatization reactions towards various high-value-added pharmaceutically relevant compounds.

Abstract

Artificial metalloenzymes (ArMs) are an attractive approach to achieving “new to nature” biocatalytic transformations. In this work, a novel copper-dependent artificial Michaelase (Cu_Michaelase) comprising a genetically encoded copper-binding ligand, i. e. (2,2-bipyridin-5-yl)alanine (BpyA), was developed. For the first time, such an ArM containing a non-canonical metal-binding amino acid was successfully optimized through directed evolution. The evolved Cu_Michaelase was applied in the copper-catalyzed asymmetric addition of 2-acetyl azaarenes to nitroalkenes, yielding various γ-nitro butyric acid derivatives, which are precursors for a range of high-value-added pharmaceutically relevant compounds, with good yields and high enantioselectivities (up to >99 % yield and 99 % ee). Additionally, the evolved variant could be further used in a preparative-scale synthesis, providing chiral products for diverse derivatizations. X-ray crystal structure analysis confirmed the binding of Cu(II) ions to the BpyA residues and showed that, in principle, there is sufficient space for the 2-acetyl azaarene substrate to coordinate. Kinetic studies showed that the increased catalytic efficiency of the evolved enzyme is due to improvements in apparent K _M for both substrates and a notable threefold increase in apparent k _cat for 2-acetyl pyridine. This work illustrates the potential of artificial metalloenzymes exploiting non-canonical metal-binding ligands for new-to-nature biocatalysis.

The nitrogen-nitrogen (N-N) bond motif comprises an important class of compounds for drug discovery. Synthetic methods are primarily based on the modification of N-N or N=N precursors, whereas selective methods for direct N-N coupling offer advantages in terms of atom economy and sustainability. In this context, enzymes such as piperazate synthases (PZSs), which naturally catalyze the N-N cyclization of L-N5-hydroxyornithine to the cyclic hydrazine L-piperazate, may allow an expansion of the current narrow range of chemical approaches for N-N coupling. In this study, we demonstrate that PZSs are able to catalyze the conversion of various N-hydroxylated diamines, which are different from the natural substrate. The N-hydroxylated diamines were obtained in situ using N-hydroxylating monooxygenases (NMOs), allowing subsequent cyclization by PZS, ultimately forming the N-N bond to yield various N-N bond-containing heterocycles. Using bioinformatic tools, we identified novel NMO and PZS homologs that exhibit distinct activity and stereoselectivity profiles. The screened panel yielded 17 hydroxylated diamines and new promiscuous NMOs, thereby expanding the substrate range of NMOs resulting in the formation of previously poorly accessible N-hydroxylated products as substrates for PZS. The subsequently investigated PZSs led to a series of 5- and 6-membered N-N bond-containing heterocycles, and the most promiscuous catalysts were used to scale up and optimize the synthesis, yielding the desired N-N bond-containing heterocycles with up to 45% isolated yield. Overall, our data provides essential insights into the substrate promiscuity and activity of NMOs and PZSs, further enhancing the potential of these biocatalysts for an expanded range of N-N coupling reactions.

Genetic incorporation of noncanonical amino acids (ncAAs) harboring catalytic side chains into proteins allows the creation of enzymes able to catalyze reactions that have no equivalent in nature. Here, we present for the first time the use of the ncAA 3-aminotyrosine (aY) as catalytic residue in a designer enzyme for iminium activation catalysis. Incorporation of aY into protein scaffold LmrR gave rise to an artificial Friedel-Crafts (FC) alkylase exhibiting complementary enantioselectivity to a previous FC-alkylase design using p-aminophenylalanine as catalytic residue in the same protein. The new FC-alkylase was optimized by directed evolution to afford a quadruple mutant that showed increased activity and excellent enantioselectivity (up to 95% ee). X-ray crystal structures of the parent and evolved designer enzymes suggest that the introduced mutations cause a narrowing of the active site and a reorientation of the catalytic -NH2 group. Furthermore, the evolved FC-alkylase was applied in whole-cell catalysis, facilitated by the straightforward incorporation of aY. Our work demonstrates that aY is a valuable addition to the biochemists toolbox for creating artificial enzymes.

In light of the ubiquity of 1,1’-disubstituted tetrahydro-ß-carboline (THBC) motif in alkaloid natural products, developing asymmetric methodology for its preparation is highly valuable. Despite the immense progress towards achieving stereoselective Pictet-Spengler reaction with aldehydes, the analogous reaction with ketones is still underdeveloped. Exploiting KslB, a Pictet-Spenglerase from the biosynthesis of kitasetaline, we develop a general, diastereoselective, and protecting-group free method for the construction of densely functionalized THBCs with α-quaternary center by coupling tryptophan derivatives and α-keto acids. We determine the stereochemistry of kitasetalic acid, KslB’s physiological product and a key biosynthetic intermediate towards kitasetaline, and established that KslB’s selectivity is opposite to what is achieved chemically. Our investigations of KslB show its high activity (TTN>438,000), substrate promiscuity, and tolerance for high substrate concentrations (0.1M). Additionally, a TrpB-KslB cascade enables the construction of complex tricyclic products from simple indoles in one-pot. X-ray structural characterization of KslB sheds light on potential active site interactions to account for its stereoselectivity and ability to accept ketone substrates.

Amino-acid residues distant from an enzyme's active site are known to influence catalysis, but their mechanistic contributions to the catalytic cycle remain poorly understood. Here, we investigate the structural, functional, and mechanistic impacts of distal and active-site mutations discovered through directed evolution of the computationally designed retro-aldolase RA95. Active-site mutations improve catalytic efficiency by 3,600-fold, while distal mutations alone offer no improvement. When combined with active-site mutations, distal mutations further increase efficiency by 6-fold, demonstrating an epistatic effect. X-ray crystallography and molecular dynamics simulations reveal that distal mutations promote active site opening by altering loop dynamics. Kinetic solvent viscosity effects and electrostatic analysis show that distal mutations accelerate the chemical transformation by 100-fold, shifting the rate-limiting step to product release, which is further accelerated by the increased opening of the active site. These findings highlight the critical role of distal residues in shaping the active-site environment and facilitating the structural dynamics essential for progression through the catalytic cycle.

Heteroaromatic alkylations are indispensable reactions for synthesizing biologically active molecules. The anti-Markovnikov hydroarylation of olefins using heteroaryl hal-ides furnishes the product as a single regioisomer, however, catalytic variants are ineffective in controlling the stereochem-ical outcome of these reactions. Here, we report a synergistic photoenzymatic hydroarylation of olefins using flavin-dependent ‘ene’-reductases with ruthenium photoredox cata-lysts. Enzyme homologs were identified, which provide access to both product enantiomers in greater than 80% yield with up to 99:1 er. This method is effective for styrenyl and unactivat-ed alkenes, highlighting the generality of this approach. Bind-ing assay study revealed strong binding of the photocatalyst with the enzyme for superior catalytic activity. Mechanistic studies suggest efficient intermolecular coupling is possible because alkene binding accelerates the consumption of the aryl halide.

Nature, Published online: 15 January 2025; doi:10.1038/s41586-024-08393-x

Deep learning methods have been used to design proteins that can neutralize the effects of three-finger toxins found in snake venom, which could lead to the development of safer and more accessible antivenom treatments.

Nature Communications, Published online: 16 January 2025; doi:10.1038/s41467-025-55987-8

Directed evolution is a powerful method to optimize protein fitness. Here, authors develop an active learning workflow using machine learning to more efficiently explore the design space of proteins.

Cooperative catalysis with an enzyme and a small-molecule photocatalyst has very recently emerged as a potentially general activation mode to advance novel biocatalytic reactions with synthetic utility. Herein, we report cooperative photobiocatalysis involving an engineered nonheme Fe enzyme and a tailored photoredox catalyst as a unifying strategy for the catalytic enantioconvergent decarboxylative azidation, thiocyanation and isocyanation of redox-active esters via a radical mechanism. Through the survey and directed evolution of nonheme Fe enzymes, we repurposed and further evolved metapyrocatechase (MPC), a nonheme Fe extradiol dioxygenase not previously studied in new-to-nature biocatalysis, for the enantioselective C–N3, C–SCN and C–NCO bond formation through a radical rebound mechanism with an enzymatic Fe–X intermediate (X = N3, NCS, and NCO). A range of primary, secondary and tertiary alkyl radical precursors were effectively converted by our engineered MPC, allowing the syntheses of organic azides, thiocyanates and isocyanates with good to excellent enantiocontrol. Further chemical derivatization of these products furnished valuable compounds including enantioenriched amines, triazoles, ureas and SCF3-containing products. Computational studies via DFT and MD simulations shed light on the mechanism as well as the binding poses of the alkyl radical intermediate in the enzyme active site and the π-facial selectivity in the enantiodetermining radical rebound. Overall, cooperative photometallobiocatalysis with nonheme Fe enzymes provides a new platform for the development of challenging asymmetric radical transformations eluding small-molecule catalysis.