R.B. Leveson-Gower

The biosynthesis of saturated bioisosteres of ortho-disubstituted benzenes was reported by the artificial photoenzyme harboring a genetic incorporated photosensor under the light with several examples after the directed evolution. Our work provides a biocatalytic strategy for the synthesis of saturated bioisosteres of ortho-disubstituted benzenes and expands the utility of artificial photoenzyme for abiological reactions.

Abstract

Saturated bioisosteres of ortho-substituted benzenes are of significant interest due to their enhanced pharmacokinetic properties, such as improved metabolic stability and reduced toxicity, making them valuable in drug design and development. However, efficient synthesis of them remains a challenge in organic chemistry. Herein, we report the biocatalytic synthesis of saturated bioisosteres of ortho-substituted benzenes using engineered artificial photoenzymes. The artificial photoenzyme, incorporating genetically encoded unnatural amino acids with benzophenone photosensitizer residue, facilitate the formation of chiral saturated bioisosteres with moderate enantiomeric excess via the energy transfer process. Our results demonstrate the versatility of artificial photoenzymes in mediating new-to-nature reactions that are difficult to achieve with conventional chemical or enzymatic methods.

A single-step biocatalytic route to complex indolizidine and quinolizidine alkaloids is described that relies on transaminase-triggered double intramolecular aza-Michael methodology. In one case, a retro-double intramolecular aza-Michael reaction enables dynamic kinetic resolution.

Abstract

Biocatalysis is now a well-established branch of catalysis and the growing toolbox of natural, evolved and designer enzymes is enabling chemistry previously deemed inaccessible. However, most enzyme methodologies have been developed for functional group interconversions, such as the conversion of a ketone into an amine or alcohol, and do not result in the generation of significant 3D molecular complexity. The application of enzyme-triggered reaction cascade methodologies has the potential to transform simple substrates into complex sp³-rich molecules in one step. Herein, we describe a single-step biocatalytic route to high-value, complex indolizidine, and quinolizidine alkaloids, which relies on a transaminase-triggered double intramolecular aza-Michael reaction. This approach allows access to architecturally complex, natural-product-like N-heterocycles and reveals intriguing examples of diastereoselectivity in these enzyme-triggered reactions. Significantly, we demonstrate an elegant example of a biocatalytic cascade where the transaminase plays a dual role in generating complex N-heterocycles and where a retro-double intramolecular aza-Michael reaction mediates a dynamic kinetic resolution and enables the isolation of sp³-rich indolizidine diastereoisomers containing five stereocenters, as single isomers.

Thioketals are an important class of compounds that enable the selective preparation and protection of carbonyl compounds in chemical synthesis. Despite their synthetic utility, selective cleavage of thioketals often requires the use of harsh conditions and stoichiometric reagents that are largely bioincompatible. Herein, we describe a biocatalytic strategy for the selective cleavage of thioketals using enzymatic bromide recycling by vanadium-dependent haloperoxidase (VHPO) enzymes. This process involves thioketal cleavage through repetitive VHPO-mediated formation of hypohalous acid with a catalytic quantity of halide salt and hydrogen peroxide as the terminal oxidant. This method is demonstrated on a broad range of 1,3-thioketals in high yield and excellent chemoselectivity. The protocol has been demonstrated on gram-scale, run with lysate and whole cells, and has been extended to cleavage of 1,3-acetals, 1,4-thioketals, and 1,3-oxathiolanes.

Nature Communications, Published online: 20 March 2025; doi:10.1038/s41467-025-58038-4

Enzyme kinetic parameter prediction is a challenge in enzyme discovery and engineering. Here, the authors train a robust deep learning model CataPro to predict enzyme kinetic parameters and validate its practicality through wet-lab experiments.

Photoenzymatic catalysis facilitates stereoselective new-to-nature chemistry under mild conditions. In addition to the rational design of artificial photoenzymes, naturally occurring redox enzymes have been repurposed for this approach. Most prominently, flavin-containing cofactors can promote photoredox catalysis in the chiral protein environment, with several examples of enantioselective C–C bond forming reactions reported in recent years. Here, we add another class of natural enzymes, which utilize the pyrroloquinoline quinone (PQQ) cofactor, to the toolbox of photobiocatalysis. Although structurally distinct from flavin, PQQ exhibits mechanistic similarities, as it also absorbs visible light and is capable of single-electron transfer. First, we established the trimethyl ester PQQMe3 as a stand-alone photoredox catalyst in pure organic solvent. Upon excitation, PQQMe3 enables the redox-neutral radical cyclization of an N-(bromoalkyl)-substituted indole. We then tested a panel of PQQ-dependent sugar and alcohol dehydrogenases for photoenzymatic catalysis in aqueous buffer, focusing on a redox-neutral radical reaction to form oxindoles. Under optimized reaction conditions, we obtained 69% yield and an 82:18 enantiomeric ratio. Our work thus demonstrates that PQQ enzymes are capable of stereoselective photoredox catalysis. Future enzyme engineering efforts based on computational modelling and directed evolution will fully unlock their synthetic potential.

The anti - and syn -selective epoxidation of various α,β-unsaturated aldehydes is promoted by an engineered variant of 2-deoxy-D-ribose-5-phosphate aldolase (DERA), giving rise to various α,β-epoxy-aldehydes with excellent enantiopurity (enantiomeric ratio up to 99 : 1).

Abstract

The enzyme 2-deoxy-D-ribose-5-phosphate aldolase (DERA) naturally catalyzes the reversible aldol addition between acetaldehyde and D-glyceraldehyde-3-phosphate to yield 2-deoxy-D-ribose-5-phosphate. Herein we describe the redesign of DERA into a proficient non-natural peroxygenase that promotes the asymmetric epoxidation of various α,β-unsaturated aldehydes. This repurposed aldolase, named DERA-EP, is able to utilize H₂O₂ to accomplish both anti- and syn-selective epoxidations of various α,β-unsaturated aldehydes to give the corresponding epoxides with moderate to high diastereoselectivity (diastereomeric ratio up to 99 : 1) and excellent enantioselectivity (enantiomeric ratio up to 99 : 1). Crystallographic analysis of DERA-EP in a substrate-free and substrate-bound state provides a structural context for the evolved activity, a clear explanation for the high enantioselectivity, and compelling evidence for catalysis via enzyme-bound iminium ion intermediates. The unprecedented anti-selectivity of DERA-EP with multiple α,β-unsaturated aldehydes is complementary to the syn-selectivity of previously reported enzyme-, metal- and organo-catalysts, making DERA-EP an attractive new asset to the toolbox of epoxidation catalysts.

As one of the most potent and selective protein phosphatase inhibitors, fostriecin shows a broad range of anticancer activity. In light of fostriecin’s antiproliferative properties, a phase I clinical trial was conducted on the natural product, but was soon halted due to issues with compound stability and purity. Numerous efforts in the past two decades have yielded 17 successful syntheses that proceed in 19 to 34 steps. Herein, we develop a modular chemoenzymatic approach that provides fostriecin and its analogs in a collective manner in 9 steps (longest linear sequence). The synthesis features a convergent assembly of three key fragments and a late-stage chemoenzymatic derivatization of an advanced intermediate that (i) installs two of the key pharmacophores and (ii) allows ready diversification of the hydrophobic tail. A key feature in this derivatization is the optimization of an enzymatic C–H oxidation step through the concurrent use of decoy molecule strategy and rational enzyme engineering. Cumulatively, our strategy capitalizes on the exquisite chemoselectivity of enzymatic transformations while ensuring synthetic modularity and versatility for analog generation. This work will facilitate future investigation into the biological activities and medicinal chemistry of the natural product family.

The role of amino-acid residues distant from an enzymes active site in facilitating the complete catalytic cycle--including substrate binding, chemical transformation, and product release-- remains poorly understood. Here, we investigate how distal mutations promote the catalytic cycle by engineering mutants of three de novo Kemp eliminases containing either active-site or distal mutations identified through directed evolution. Kinetic analyses, X-ray crystallography, and molecular dynamics simulations reveal that while active-site mutations create preorganized catalytic sites for efficient chemical transformation, distal mutations enhance catalysis by facilitating substrate binding and product release through tuning structural dynamics to widen the active-site entrance and reorganize surface loops. These distinct contributions work synergistically to improve overall activity, demonstrating that a well-organized active site, though necessary, is not sufficient for optimal catalysis. Our findings reveal critical roles that distal residues play in shaping the catalytic cycle to enhance efficiency, yielding valuable insights for enzyme design.

Biocatalysis is a branch of catalysis that has allowed the development of a diverse range of sophisticated synthetic methodologies that target geometrically demanding structures such as pharmaceuticals, natural products, and their analogues. The routes are often more efficient due to enzymes innately high degree of chemo-, regio- and stereoselectivity, while making the overall process more sustainable when compared to their chemical equivalent, as enzymes are naturally biodegradable and operate under physiological conditions. Herein, we demonstrate the power of this catalytic approach via the development of a chemoenzymatic one-pot process that allowed access to the fungal metabolites tenuipesones A and B, along with their enantiomeric counterparts, in good yields (60-72 %) and excellent enantioselectivity (>99 % ee). The stereochemical outcome of the products was controlled through careful selection of the biocatalysts, enabling control of the configuration of the key C7 chiral center. This novel chemoenzymatic cascade process allowed access to all four possible stereoisomers, with their absolute stereochemistry being evaluated to confirm the true configuration of the natural isolates.

Proceedings of the National Academy of Sciences, Volume 122, Issue 14, April 2025.
SignificanceA fundamental concept in chemistry is that the properties of elements are periodic. Magnesium is a ubiquitous alkaline Earth (group II element) metal cofactor in enzymes that catalyzes many biochemical reactions, including DNA processing. We ...

De novo enzyme design starts from ideal active site descriptions consisting of constellations of catalytic residue functional groups around reaction transition state(s), and seeks to generate protein structures that can accurately hold the site in place. Highly active enzymes have been designed starting from such descriptions using the generative AI method RFdiffusion [1-3], but there are two current methodological limitations. First, the geometry of the active site can only be specified at the residue level, so for each catalytic residue functional group placed around the reaction transition state, the possible locations of the residue backbone must be enumerated by building side chain rotamers back from the functional group. Second, the location of the catalytic residues along the sequence must be specified in advance, which considerably limits the space of solutions which can be sampled. Here we describe a new deep generative method, Rosetta Fold diffusion 2 (RFdiffusion2), that solves both problems, enabling enzymes to be designed from sequence agnostic descriptions of functional group locations without inverse rotamer generation. We first evaluate RFdiffusion2 on an in silico enzyme design benchmark of 41 diverse active sites and find that it is able to successfully build proteins scaffolding all 41 sites, compared to 16/41 with prior state-of-the-art deep learning methods. Next, we design enzymes around three diverse catalytic sites and characterize the designs experimentally; in each case we identify active catalysts in testing less than 96 sequences. RFdiffusion2 demonstrates the potential of atomic resolution generative models for the design of de novo enzymes directly from their reaction mechanisms.

De novo protein design has been used to create functional protein assemblies, but its reliance on standard amino acids limits the integration of synthetic supramolecular strategies for precise control of molecular assemblies. Herein, we present an artificial protein assembly that integrates synthetic supramolecular design with de novo protein engineering. Focusing on unit and connection rigidity, we designed and created the Bi-Porphyrin Acquisition Designer protein (BiPAD), which captures two highly designable synthetic porphyrins, via state-of-the-art generative protein design to fuse α/β-folded porphyrin-binding motifs. BiPADs captured rigid porphyrins, each bearing an additional metal coordination site, resulting in a metal-responsive cyclic assembly with the intended structure. Furthermore, high-speed atomic force microscopy revealed dynamic structural changes in the BiPAD assembly. This work expands the designability of artificial protein assemblies, paving the way for the synergistic design of functional systems through the integration of protein engineering and synthetic chemistry.

Science, Volume 388, Issue 6743, Page 142-142, April 2025.

A new-to-nature biocatalytic radical alkylation of 2-acyl imidazoles is achieved through the cross-integration of a Lewis-acid (LA)-type artificial metalloenzyme (ArM) and a photobiocatalysis strategy. Directed evolution leads to enantiodivergent synthesis with different mutants. Detailed mechanistic studies illustrate a difference in reactivity and enantiomeric preference between the illuminated and dark conditions.

Abstract

Artificial metalloenzymes (ArMs) and photoenzymatic catalysis represent two cutting-edge approaches to creating new enzyme reactivity. However, the potential of merging these two strategies remains underdeveloped for enantiocontrolled biotransformations. Herein, we develop a synergistic metalloenzymatic and photoredox catalysis platform to enable enantiodivergent radical alkylation of 2-acyl imidazoles. Specifically, cupin proteins are redesigned to function as copper(II)-based Lewis-acid-enzymes (LAses), which, in synergy with tripyridinyl-ruthenium-based photoredox catalysis, precisely control the generation, reactivity, and selectivity of abiological radicals, thereby unlocking non-natural enzyme reactivity. Powered by protein engineering, repurposed photo-LAses facilitate the green and efficient synthesis of diverse enantioenriched α-chiral ketones in high enantioselectivity (both enantiomers accessible, up to 97% yield and 98.5:1.5 enantiomeric ratio [er]). Detailed mechanistic studies suggest a radical addition to the metalloenzymatic enolate pathway and explain the switched selectivity from dark to photoconditions.

We explore KslB, a Pictet–Spenglerase (PSase) capable of accepting ketones, in preparing 1,1′-disubstituted-ß-carbolines. Biocatalytic optimizations show its broad promiscuity, excellent stereoselectivity, stability (TTN > 4 38 000), and application in an enzyme-cascade with tryptophan synthase. X-ray characterization demonstrate conserved features among bacterial PSases like McbB and KslB and offered insights into KslB's ability to accept ketones.

Abstract

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 toward 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 toward kitasetaline, and established that KslB's selectivity is opposite to what is achieved chemically. Our investigations of KslB show its high activity (total turnover number >438000), substrate promiscuity, and tolerance for high substrate concentrations (0.1 M). 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.

Nature Synthesis, Published online: 14 April 2025; doi:10.1038/s44160-025-00788-6

A repurposed non-haem, iron-based dioxygenase enables the stereoconvergent reduction of alkenes with excellent selectivity. Mechanistic studies support an iron hydride pathway and reveal the molecular mechanism of stereoconvergence.

Enzymes achieve catalysis by dynamically sampling diverse conformational states. Beyond this plasticity, individual enzyme molecules occupy metastable substates, forming an ensemble of functional substates within a population. Since shifts in functional substate dynamics drive phenotypic variation, their evolutionary trajectories are central to the emergence of new functions. However, the challenge of measuring functional substates has hindered our understanding of their role in enzyme evolution and the optimization of conformational substates. Here, we address this gap by investigating how functional and conformational substates were modulated during enzyme functional transitions, using single-molecule kinetic assays and molecular dynamics simulations. We analyzed wild-type phosphotriesterase (PTE) and 18 evolved variants that transitioned from the native PTE to promiscuous arylesterase (AE) activity. Our findings reveal that evolutionary transitions reshape functional and conformational substate landscapes: PTE-specialized variants exhibit broader substate distributions, whereas AE-specialized variants display more uniform substates. These results provide the first direct evidence that enzyme evolution is accompanied by coordinated shifts in functional and conformational substate equilibria, optimizing both for the enzymes catalytic efficiency. This work highlights the power of single-molecule techniques in uncovering how heterogeneous enzyme populations navigate substate transitions and, ultimately, how these transitions shape enzyme evolvability.

Biocatalysis has become a sustainable and cost-competitive alternative to established chemical synthesis, enabling the enzyme-based production of not only commodity chemicals but (non-natural) amino acids, (rare) sugars, as well as synthetic nucleotides. These building blocks give access to highly complex molecules with versatile industrial and therapeutic applications.

Abstract

Supported by rapid technological advancements, biocatalytic applications have matured into sustainable, scalable, and cost-competitive alternatives to established chemical catalysis. This review presents the most recent examples of enzyme-based solutions for the manufacturing of molecules with extended carbon–carbon frameworks and multiple stereogenic centers at commercial scale, including peptide building blocks, (rare) sugars, synthetic (oligo)nucleotides, and terpenoids, such as (–)-Ambrox^®. Novel enzyme classes are highlighted along with their potential applications—the synthesis of DNA/RNA, the depolymerization of synthetic plastics, or fully enzymatic protection/deprotection schemes—pointing toward the diversification and broader industrial utilization of biocatalysis-based processes.

Innovation in biocatalysis is rapidly increasingly the diversity of catalytic reactivity that can be mediated by enzymes, addressing a key bottleneck for their widespread adoption in industrial chemical synthesis. A key approach to this is building enzymes with unnatural catalytic components that provide an expanded palette with new possibilities for enzymatic reactivity.

The expanding applications of biocatalysis in the chemical and pharmaceutical sectors herald a greener future for these industries. Yet, the range of chemical reactions known to enzymes only covers a small fraction of what is required for modern synthetic routes. To continue the increases in sustainability afforded by converting chemical processes into enzymatic ones, fundamentally new kinds of biocatalytic reactivity are required. Perhaps the very components from which enzymes are constructed, a palette of canonical amino acids and cofactors, inherently limit their catalytic possibilities, even if all the available natural sequence space can be explored. In recent years, there has been an explosion of strategies to produce new biocatalytic function through the incorporation of noncanonical amino acids and synthetic cofactors, new colors which are added to the enzyme design palette. This has enabled new enzymatic reactions that proceed via organocatalytic, organometallic, and photocatalytic mechanisms. Aside from designing new enzymatic activities from scratch, exogenous photocatalysts have recently also been used in synergy with natural enzyme active sites to diverge their reactivity towards radical pathways. This review will highlight recent developments in enriching enzymatic chemistry with new unnatural components, providing an outlook for future directions and needed developments for practicality and sustainability.

An efficient biocatalytic platform has been established for the synthesis of cis/trans and axial chiral cyclohexylamines with excellent stereoselectivity, high yield, and a broad substrate scope. The industrial applicability of this biocatalytic approach was exemplified through 10g-scale preparative reactions and the chemo-enzymatic synthesis of key intermediates for the 11β-HSD1 inhibitors AM-7715.

Abstract

Functionalizing the symmetric carbonyl carbon of cyclohexanone to achieve stereocontrol, resulting in saturated cyclohexanes with either cis/trans or axial stereochemistry, poses significant challenges in chemical synthesis, and existing methodologies are limited. In this study, we present an enzymatic reductive amination strategy to attain this objective. By engineering the enzyme pocket of the sole imine reductase (IRED) M5, we successfully synthesized over 80 cis/trans and axially chiral 4-substituted cyclohexylamines in a stereo-complementary fashion, adhering to industrial standards, via the reductive amination of 4-substituted cyclohexanones. Mechanistically, the reshaping of the enzyme pocket allows the optimized variants to distinguish between different imine precursors and selectively bind their specific configurations with favorable binding energies, thereby facilitating the generation of stereochemically distinct products. We propose that this stereocontrolled-functionalization strategy could be extended to a broader range of cyclohexylamines with diverse substituents.