Nature Catalysis, Published online: 29 May 2024; doi:10.1038/s41929-024-01176-7
A boronic enzymeR.B. Leveson-Gower
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Order matters in evolution
Nature Catalysis, Published online: 29 May 2024; doi:10.1038/s41929-024-01163-y
The understanding of protein evolution is a central challenge in biology. Now, the evolution of a β-lactamase in vitro reveals that the total effect of mutations can change the rate-limiting step of the catalytic mechanism.Design and Evolution of an Enzyme for the Asymmetric Michael Addition of Cyclic Ketones to Nitroolefins by Enamine Catalysis
Highly stereoselective Michael addition of cyclic ketones to nitroolefins was promoted by a designer artificial enzyme harboring a catalytic pyrrolidine residue through enamine catalysis. Diverse chiral γ-nitroketones were prepared by this efficient biocatalytic strategy for ketone functionalization in a study highlighting the utility of artificial enzymes for new-to-nature reactions.
Abstract
Consistent introduction of novel enzymes is required for developing efficient biocatalysts for challenging biotransformations. Absorbing catalytic modes from organocatalysis may be fruitful for designing new-to-nature enzymes with novel functions. Herein we report a newly designed artificial enzyme harboring a catalytic pyrrolidine residue that catalyzes the asymmetric Michael addition of cyclic ketones to nitroolefins through enamine activation with high efficiency. Diverse chiral γ-nitro cyclic ketones with two stereocenters were efficiently prepared with excellent stereoselectivity (up to 97 % e.e., >20 : 1 d.r.) and good yield (up to 86 %). This work provides an efficient biocatalytic strategy for cyclic ketone functionalization, and highlights the usefulness of artificial enzymes for extending biocatalysis to further non-natural reactions.
Decoding the brain
The β-subunit of tryptophan synthase is a latent tyrosine synthase
R.B. Leveson-Gowernice to see this one finally out
Nature Chemical Biology, Published online: 14 May 2024; doi:10.1038/s41589-024-01619-z
Biochemical pathways for aromatic amino acid synthesis are ancient and highly conserved. Directed evolution of the β-subunit of tryptophan synthase (TrpB)—a proficient biocatalyst that converts indole to l-tryptophan—enabled this enzyme to make l-tyrosines from phenols, a pathway not (yet) known in nature.Boron catalysis in a designer enzyme
Nature, Published online: 08 May 2024; doi:10.1038/s41586-024-07391-3
A completely genetically encoded boronic-acid-containing designer enzyme was created and characterized using X-ray crystallography, high-resolution mass spectrometry and 11B NMR spectroscopy, allowing chemistry that is unknown in nature and currently not possible with small-molecule catalysts.A deconstruction-reconstruction strategy for pyrimidine diversification
Nature, Published online: 02 May 2024; doi:10.1038/s41586-024-07474-1
A deconstruction-reconstruction strategy for pyrimidine diversificationSynergistic Photoenzymatic Catalysis Enables Synthesis of a-Tertiary Amino Acids Using Threonine Aldolases
Journals investigating Nobel winner’s papers
Biocatalytic, enantioenriched primary amination of tertiary C–H bonds
Nature Catalysis, Published online: 03 May 2024; doi:10.1038/s41929-024-01149-w
Direct stereoselective amination of tertiary C–H bonds without the assistance of directing groups is a challenging task in synthetic organic chemistry. Now a nitrene transferase is engineered to aminate tertiary C–H bonds with high enantioselectivity, providing direct access to valuable chiral α-tertiary primary amines.Biocatalytic enantioselective C(sp3)–H fluorination enabled by directed evolution of non-haem iron enzymes
Nature Synthesis, Published online: 26 April 2024; doi:10.1038/s44160-024-00536-2
Development of fluorine rebound processes at an enzymatic Fe(III) centre are a challenge. Now, a plant-derived non-haem iron enzyme, 1-aminocyclopropane-1-carboxylic acid oxidase, is repurposed and evolved to catalyse chemo- and enantioselective C(sp3)–H fluorination, forming a range of enantioenriched organofluorine products.Directed Evolution of an Artificial Hydroxylase Based on a Thermostable Human Carbonic Anhydrase Protein
Biocatalytic Oxidative Amination of para‐Substituted Phenols
Vanillyl alcohol oxidases (VAOs) are catalytically promiscuous oxidases acting on para-substituted phenols. Engineering of a VAO-type biocatalyst permits a new chemoenzymatic reaction - release of and nucleophilic addition to a reactive electrophilic intermediate, yielding α-aminated and -thiolated para-alkylphenols.
Abstract
Biocatalytic preparation of chiral amines is a large and burgeoning field in organic chemistry. Many enzymes and routes have been published, including transaminases, imine reductases, reductive aminases, amine dehydrogenases and others. However, all these routes rely on some sacrificial substrate, in the form of either amine donor or cofactor regeneration substrate. Herein, we report the direct oxidative amination of p-substituted phenols catalyzed by an evolved flavoprotein oxidase, with the consumption of only substrate and O2, and release of H2O2. The substrate scope of the reaction is studied, and is tolerant of a diverse panel including ammonia, primary and secondary amines, and amino acids. The reaction is later employed at preparative scale to generate aminated products in 50–80 % yield. This report establishes flavoprotein oxidase as a new and economical member of the chemist's toolkit for biocatalytic generation of chiral amines, acting as oxidative aminase.
Artificial Metalloenzyme‐Catalyzed Enantioselective Carboamination of Alkenes
An artificial metalloenzyme (ArM) based on biotin-streptavidin technology was repurposed for enantioselective nonannulative carboamination of alkenes. The combination of design of experiment (DoE) and genetic optimization led to a >630 % improvement in turnover number (TON).
Abstract
Relying on ubiquitous alkenes, carboamination reactions enable the difunctionalization of the double bond by the concurrent formation of a C−N and a C−C single bond. In the past years, several groups have reported on elegant strategies for the carboamination of alkenes relying on homogeneous catalysts or enzymes. Herein, we report on an artificial metalloenzyme for the enantioselective carboamination of dihydrofuran. Genetic optimization, combined with a Bayesian optimization of catalytic performance, afforded the disubstituted tetrahydrofuran product in up to 22 TON and 85 % ee. X-ray analysis of the evolved artificial carboaminase shed light on critical amino acid residues that affect catalytic performance.
[ASAP] Photoenzymatic Redox-Neutral Radical Hydrosulfonylation Initiated by FMN
Stereodivergent photobiocatalytic radical cyclization through the repurposing and directed evolution of fatty acid photodecarboxylases
R.B. Leveson-Gowerhe just keeps droppin em
Nature Chemistry, Published online: 17 April 2024; doi:10.1038/s41557-024-01494-0
Despite their intriguing photochemical activities, natural photoenzymes have not yet been repurposed for new-to-nature activities. Now, by leveraging the strongly oxidizing excited-state flavoquinone cofactor, fatty acid photodecarboxylases were engineered to catalyse unnatural decarboxylative radical cyclization with excellent chemo-, enantio- and diastereoselectivities.Artificial manganese metalloenzymes with laccase-like activity: Design, Synthesis and Characterization
Unnatural Thiamine Radical Enzymes for Photobiocatalytic Asymmetric Alkylation of Benzaldehydes and a-Ketoacids
On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non‐Native Halogenases
Electrophilic halogenases in nature are typically not efficient. Guided by flavin-dependent halogenase mechanisms and taking advantage of the versatile reactivity of a flavin hydroperoxide adduct and in situ generation of H2O2 by flavin-dependent enzymes, it was possible to promote the formation of a hypohalous acid—which is key for electrophilic halogenation—in various non-native halogenases by rerouting the flavin-generated peroxide.
Abstract
Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (FlC4a-OOH), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H2O2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that FlC4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from FlC4a-OOH cannot halogenate their substrates. Remarkably, in situ H2O2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming FlC4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation.
[ASAP] Practical Machine Learning-Assisted Design Protocol for Protein Engineering: Transaminase Engineering for the Conversion of Bulky Substrates
[ASAP] From Ground-State to Excited-State Activation Modes: Flavin-Dependent “Ene”-Reductases Catalyzed Non-natural Radical Reactions
Emergence of fractal geometries in the evolution of a metabolic enzyme
Nature, Published online: 10 April 2024; doi:10.1038/s41586-024-07287-2
Citrate synthase from the cyanobacterium Synechococcus elongatus is shown to self-assemble into Sierpiński triangles, a finding that opens up the possibility that other naturally occurring molecular-scale fractals exist.Modern approaches to therapeutic oligonucleotide manufacturing
Closed-loop recyclability of a biomass-derived epoxy-amine thermoset by methanolysis
An Artificial Enzyme for Asymmetric Nitrocyclopropanation of α,β‐Unsaturated Aldehydes—Design and Evolution
R.B. Leveson-GowerInteresting that they chose a scaffold with a catalytic lysine for this.
Generation of an artificial enzyme that features a secondary amine residue by genetic code expansion is described. The designer enzyme was evolved to catalyze the asymmetric nitrocyclopropanation of cinnamaldehydes at high conversions with excellent diastereo- and enantioselectivity.
Abstract
The introduction of an abiological catalytic group into the binding pocket of a protein host allows for the expansion of enzyme chemistries. Here, we report the generation of an artificial enzyme by genetic encoding of a non-canonical amino acid that contains a secondary amine side chain. The non-canonical amino acid and the binding pocket function synergistically to catalyze the asymmetric nitrocyclopropanation of α,β-unsaturated aldehydes by the iminium activation mechanism. The designer enzyme was evolved to an optimal variant that catalyzes the reaction at high conversions with high diastereo- and enantioselectivity. This work demonstrates the application of genetic code expansion in enzyme design and expands the scope of enzyme-catalyzed abiological reactions.
[ASAP] A Proline-Based Artificial Enzyme That Favors Aldol Condensation Enables Facile Synthesis of Aliphatic Ketones via Tandem Catalysis
R.B. Leveson-Gowerstretching the definition of artificial enzymes once again
Photocatalytic Functionalization of Dehydroalanine‐Derived Peptides in Batch and Flow
A photocatalytic hydroarylation of dehydroalanine (Dha) and Dha-containing peptides with versatile arylthianthrenium salts was developed in batch and in flow, enabling expedient scale-up. The mild nature of the photocatalytic approach allowed the diversification of peptides featuring various sensitive functional groups and the effective stitching of Dha-containing peptides with a myriad of arenes and drug scaffolds.
Abstract
Unnatural amino acids, and their synthesis by the late-stage functionalization (LSF) of peptides, play a crucial role in areas such as drug design and discovery. Historically, the LSF of biomolecules has predominantly utilized traditional synthetic methodologies that exploit nucleophilic residues, such as cysteine, lysine or tyrosine. Herein, we present a photocatalytic hydroarylation process targeting the electrophilic residue dehydroalanine (Dha). This residue possesses an α,β-unsaturated moiety and can be combined with various arylthianthrenium salts, both in batch and flow reactors. Notably, the flow setup proved instrumental for efficient scale-up, paving the way for the synthesis of unnatural amino acids and peptides in substantial quantities. Our photocatalytic approach, being inherently mild, permits the diversification of peptides even when they contain sensitive functional groups. The readily available arylthianthrenium salts facilitate the seamless integration of Dha-containing peptides with a wide range of arenes, drug blueprints, and natural products, culminating in the creation of unconventional phenylalanine derivatives. The synergistic effect of the high functional group tolerance and the modular characteristic of the aryl electrophile enables efficient peptide conjugation and ligation in both batch and flow conditions.
Theoretical Insights into the Reduction of Azurin Metal Site with Unnatural Amino Acid Substitutions
Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl‐Alkenes Catalyzed by Laboratory Evolved P450s
Computational modelling involving density functional theory (DFT) calculations, molecular dynamics (MD) simulations, and hybrid quantum mechanics / molecular mechanics (QM/MM) calculations, are used to investigate and decipher the mechanism for chemoselectivity control achieved by a set of laboratory evolved P450s for selective allylic C−H hydroxylation vs. epoxidation and carbonyl formation of internal aryl-alkenes.
Abstract
P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C−H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-β-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C−H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.