Braca

Recent advancements in the preparation of immobilized enzymes have improved both batch and continuous flow biocatalytic processes, essential for cost-effective, sustainable production in the fine chemicals and pharmaceutical industries. This review examines advancements in carrier-free methods, entrapment strategies, and support-based approaches, discusses material choices, and explores novel binding techniques like genetic fusion for better enzyme activity and stability.

Abstract

The development of immobilized enzymes both for batch and continuous flow biocatalytic processes has gained significant traction in recent years, driven by the need for cost-effective and sustainable production methods in the fine chemicals and pharmaceutical industries. Enzyme immobilization not only enables the recycling of biocatalysts but also streamlines downstream processing, significantly reducing the cost and environmental impact of biotransformations. This review explores recent advancements in enzyme immobilization techniques, covering both carrier-free methods, entrapment strategies and support-based approaches. At this regard, the selection of suitable materials for enzyme immobilization is examined, highlighting the advantages and challenges associated with inorganic, natural, and synthetic organic carriers. Novel opportunities coming from innovative binding strategies, such as genetic fusion technologies, for the preparation of heterogeneous biocatalysts with enhanced activity and stability will be discussed as well. This review underscores the need for ongoing research to address current limitations and optimize immobilization strategies for industrial applications.

This review summarizes the functional, structural, and mechanistic properties of flavin-dependent nitroreductases and highlights their usefulness for the synthesis of nitrogen-containing compounds. Recent advances in enzyme and cofactor engineering, as well as in the use of nitroreductases in photobiocatalytic approaches, are emphasized. The versatility and promiscuity of nitroreductases and related flavoenzymes holds great potential to develop new enzyme reactivities.

Abstract

Nitroreductases, present within both prokaryotes and eukaryotes, form a group of flavin-dependent enzymes capable of reducing nitro compounds using NAD(P)H as reducing agent. These enzymes have been widely studied due to their diverse roles in bioremediation, cancer therapy, cell ablation, and antimicrobial resistance. In recent times, the versatility of nitroreductases has been expanded toward the synthesis of highly valuable compounds such as aromatic and aliphatic amines, azoxy and azobenzenes, as well as N-heterocycles. This review examines the biological role and diversity of flavin-dependent nitroreductases, and highlights their current and potential future application as biocatalysts for the sustainable synthesis of nitrogen-containing pharmaceutical compounds and bulk chemicals.

The Nobel Prize for Chemistry 2024 was jointly awarded to David Baker for computational protein design and to Demis Hassabis and John Jumper for protein structure prediction. This highlight showcases the impact of the Nobel prize laureates’ contributions and summarizes the history, state of the art, applications and future directions of these methods.

Abstract

The ability to predict and design protein structures has led to numerous applications in medicine, diagnostics and sustainable chemical manufacture. In addition, the wealth of predicted protein structures has advanced our understanding of how life's molecules function and interact. Honouring the work that has fundamentally changed the way scientists research and engineer proteins, the Nobel Prize in Chemistry in 2024 was awarded to David Baker for computational protein design and jointly to Demis Hassabis and John Jumper, who developed AlphaFold for machine-learning-based protein structure prediction. Here, we highlight notable contributions to the development of these computational tools and their importance for the design of functional proteins that are applied in organic synthesis. Notably, both technologies have the potential to impact drug discovery as any therapeutic protein target can now be modelled, allowing the de novo design of peptide binders and the identification of small molecule ligands through in silico docking of large compound libraries. Looking ahead, we highlight future research directions in protein engineering, medicinal chemistry and material design that are enabled by this transformative shift in protein science.

A new class of artificial metalloenzymes containing genetically encoded noble-metal-binding sites featuring a non-canonical thiophenol-based amino acid, which serves as an excellent soft ligand for binding various noble metals, was developed. The corresponding gold(I) enzyme was characterised, confirming gold binding to the thiophenol, and successfully applied in catalytic hydroamination reactions.

Abstract

Incorporating noble metals in artificial metalloenzymes (ArMs) is challenging due to the lack of suitable soft coordinating ligands among natural amino acids. We present a new class of ArMs featuring a genetically encoded noble-metal-binding site based on a non-canonical thiophenol-based amino acid, 4-mercaptophenylalanine (pSHF), incorporated in the transcriptional regulator LmrR through stop codon suppression. We demonstrate that pSHF is an excellent ligand for noble metals in their low oxidation states. The corresponding gold(I) enzyme was characterised by mass spectrometry, UV/Vis spectroscopy and X-ray crystallography and successfully catalysed hydroamination reactions of 2-ethynyl anilines with turnover numbers over 50. Interestingly, two equivalents of gold(I) per protein dimer proved to be required for activity. Up to 98 % regioselectivity in the hydroamination of an ethynylphenylurea substrate was observed, yielding the corresponding phenyl-dihydroquinazolinone product, consistent with a π-activation mechanism by single gold centres. The ArM was optimized by site saturation mutagenesis using an on-bead screening protocol. This resulted in a single mutant that showed higher activity for one class of substrates. This work brings the power of noble-metal catalysis into the realm of enzyme engineering and establishes thiophenols as alternative ligands for noble metals, providing new opportunities in coordination chemistry and catalysis.

By integrating enzymatic catalysis with photocatalysis, photoenzymatic catalysis emerges as a powerful strategy to enhance enzyme catalytic capabilities and provide superior stereocontrol in reactions involving reactive intermediates. Repurposing naturally occurring enzymes using visible light is among the most active directions of photoenzymatic catalysis. This Minireview focuses on a cutting-edge strategy in this direction, namely single-electron-oxidation triggered non-natural biotransformations catalyzed by photoexcited enzymes. These straightforward transformations feature a unique radical mechanism initiated by single-electron-oxidation, achieving redox-neutral non-natural C-C, C-O, and C-S bond formations, and expanding the chemical toolbox of enzymes. By highlighting recent advances in this field and emphasizing their catalytic mechanisms and synthetic potentials, innovative approaches for further photobiomanufacturing are anticipated.

Nature Chemistry, Published online: 28 November 2024; doi:10.1038/s41557-024-01675-x

The toolbox of artificial fluorescent proteins can be expanded by engineering mimics of the molecular rotor-based fluorophore found in the green fluorescent protein (GFP) into diverse protein scaffolds. Now, by genetically encoding mimics of the GFP fluorophore, any protein of interest can be modified to fluoresce either under select circumstances or always (when folded).

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) was created that comprises a genetically encoded copper-binding ligand, i.e. (2,2-bipyridin-5-yl)alanine (BpyA). For the first time, such an ArM containing a non-canonical metal-binding amino acid was successfully optimized through directed evolution. The ArM was applied in the copper-catalyzed asymmetric Michael addition of 2-acetyl azaarenes to nitroalkenes, yielding various γ-nitro butyric acid derivatives, which are precursors for a range high-value-added pharmaceutically relevant compounds, with good yields and high enantioselectivities (up to >99% yield and 99% ee). The evolved ArM could even be used in a preparative scale synthesis and the products were further derivatized. X-ray crystal structure analysis confirms the binding of the Cu(II) ions to the BpyA residues and shows that, in principle, there is sufficient space for the 2-acetyl azaarene substrates to coordinate. Kinetic studies showed that the increased catalytic efficiency of the evolved enzyme is due to improvements in apparent KM for both substrates and a notable threefold increase in apparent kcat for the 2-acetyl azaarene. This work illustrates the potential of artificial metalloenzymes exploiting non-canonical metal-binding ligands for new-to-nature biocatalysis.

Nature Chemistry, Published online: 26 November 2024; doi:10.1038/s41557-024-01671-1

Expanding the biocatalysis toolbox for selective desaturation is of great value. Now ‘ene’-reductases have been repurposed to mediate dehydrogenation, the reverse process of their native activity. The developed biocatalytic desaturation platform enables desymmetrizing desaturation of cyclohexanones for the synthesis of diverse cyclohexenones that bear a remote quaternary stereogenic centre.

Nature, Published online: 21 November 2024; doi:10.1038/s41586-024-08399-5

Synergistic photobiocatalysis for enantioselective triple radical sorting

Nature, Published online: 19 November 2024; doi:10.1038/d41586-024-03746-y

The largest scholarly search engine is celebrating its 20th birthday, but AI-driven competitors offer advantages.

Nature, Published online: 20 November 2024; doi:10.1038/s41586-024-08327-7

Photocatalytic C–F bond activation in small molecules and polyfluoroalkyl substances

Introducing an arginine (Arg) residue adjacent to the latent bioreactive unnatural amino acid (Uaa) significantly accelerates both SuFEx and PFEx reaction rates between proteins. This offers a general and biocompatible strategy to harness these robust click chemistries for fundamental and applied biological research, applicable in vitro and in vivo.

Abstract

Sulfur fluoride exchange (SuFEx) and phosphorus fluoride exchange (PFEx) click chemistries are advancing research across multiple disciplines. By genetically incorporating latent bioreactive unnatural amino acids (Uaas), these chemistries have been integrated into proteins, enabling precise covalent linkages with biological macromolecules and paving the way for new applications. However, their suboptimal reaction rates in proteins limit effectiveness, and traditional catalytic methods for small molecules are often incompatible with biological systems or in vivo applications. We demonstrated that introducing an arginine adjacent to the latent bioreactive Uaa significantly boosts SuFEx and PFEx reaction rates between proteins. This method is effective across various Uaas, target residues, and protein environments. Notably, it also enables efficient SuFEx reactions in acidic conditions, common in certain cellular compartments and tumor microenvironments, which typically hinder SuFEx reactions. Furthermore, we developed the first covalent cell engager that substantially enhances natural killer cell activation through improved covalent interaction facilitated by arginine. These findings provide mechanistic insights and offer a biocompatible strategy to harness these robust chemistries for advancing biological research and developing new biotherapeutics.

Synthesis of substituted anilines upon nucleophilic addition of secondary amines to cyclohexanone derivatives followed by aromatization of the enamine by employing a combination of Ir-polypyridine complex as a photoredox catalyst and cobaloxime as H2-evolution catalyst was developed recently by Leonori et al. In this work, we replace homogeneous photoredox catalyst by heterogeneous mesoporous graphitic carbon nitride (mpg-CN) that is free of rare elements. Substituted aromatic amine and H2 are formed simultaneously. Combination of X-Ray spectroscopies revealed charge transfer from cobaloxime to mpg-CN in the dark. Illumination of the catalytic system with visible light induces electron transfer from mpg-CN to cobaloxime and formation of persistent Co(II) species. The results of DFT modelling suggest that the studied reaction is strongly endothermic and endergonic. Thus, energy of photons is stored in the reaction products – H2 and the aromatic amine.

Nature, Published online: 11 November 2024; doi:10.1038/d41586-024-03708-4

The code underlying the Nobel-prize-winning tool for modelling protein structures can now be downloaded by academics.

Biocatalysts are prized for their selectivity, tunability, and their compatibility with environmentally-friendly reaction conditions. Introduction of unnatural cofactors opens the door to new reactive enzymatic intermediates, and in turn, the possibility for new biochemical reactions. In the present study, we employed a de novo biosynthesized, non-natural cofactor, cobalt protoporphyrin IX,1 to generate a mono-nuclear cobalt hydride intermediate in the active site of a common P450 scaffold. We show that this cobalt hydride intermediate engages in metal-hydrogen atom transfer (M-HAT) reactivity, a well-studied and highly utilized reactivity pattern in synthetic chemistry,2 but which is not known to operate in nature. Because the required cofactor is fully biosynthesized and incorporated into proteins in vivo, the catalysts are highly amendable to directed evolution. We leveraged the ability to quickly access these new artificial metalloenzymes with a colorimetric screen and evolved new variants for M-HAT-mediated deallylation of phenols. We showed how common silanes have a propensity to hydrolysis that can be overcome with directed evolution by accelerating metal-hydride formation from a bulky, water stable silane. During this evolution, we discovered that variants were catalyzing HAT to the colorimetric probe itself, resulting in a unique reductive dearomatization reaction. This radical process occurs efficiently under aerobic conditions and reactions of this type have not been observed previously. These discoveries demonstrate how the tunability of biocatalytic systems can enable innovations in synthetic chemistry. We anticipate that further engineering and study of M-HAT biocatalysts will prompt new questions about hydrogen atom transfer reactivity and enable the adoption of biocatalysts for numerous synthetically useful transformations.

Transition metal–hydrides have been widely exploited in homogenous catalysis for hydrofunctionalization of unsaturated moieties, including carbonyls, alkenes and alkynes. As a complement to the well-established chemistry of these complexes involving heterolytic metal–hydride bond cleavage, metal–hydride hydrogen atom transfer (MHAT) has attracted increased interest, as it offers a promising strategy for radical hydrofunctionalziation of unactivated alkenes thus enabling late-stage diversification of complex molecules. However, due the weak interactions between the prochiral organic radical species and the enantiopure metal catalyst, achieving asymmetric MHAT remains challenging. Herein, we report our efforts to repurpose cytochrome P450 enzymes to catalyze asymmetric MHAT, a new-to-nature reaction. Directed evolution of the well-studied P450BM3 (CYP102A1) enzyme led to the identification of a triple mutant that catalyzes asymmetric MHAT radical cyclization of unactivated alkenes to afford diverse cyclic compounds, including pyrrolidines, in up to a 97:3 enantiomeric ratio under aerobic whole cell conditions. Mechanistic investigations support an MHAT mechanism proceeding via homolytic cleavage of a fleeting iron(III)hydride species. Directed evolution using CYP119 as hemoprotein scaffold led to the identification of a stereocomplementary MHATase, highlighting the potential of repurposed hemoproteins for MHAT biocatalysis. Our study showcases the potential of integrating abiotic metal–hydride activity into native metalloenzymes to expand the scope of asymmetric radical biocatalysis.

When nature evolves a gene over eons at scale, it produces a diversity of homologous sequences with patterns of conservation and change that contain rich structural, functional, and historical information about the gene. However, natural gene diversity ...