Biocatalysis@TUDelft

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.

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.

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.

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.

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

Engineered enzymes for enantioselective nucleophilic aromatic substitutions

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.

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 ...

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.

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.

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.

Nature Catalysis, Published online: 20 December 2024; doi:10.1038/s41929-024-01263-9

Photoredox catalysis is merged with metalloenzymatic catalysis to enable asymmetric decarboxylative azidation and thiocyanation. These transformations are achieved by coupling the photoredox activation of N-hydroxyphthalimide esters using a synthetic photocatalyst with enantioselective radical capture by Fe(iii) intermediates of non-haem iron enzymes.

Science, Volume 386, Issue 6728, Page 1421-1427, December 2024.

Mn(salen)-based artificial metalloenzymes (ArMs) were constructed by embedding biotinylated Mn(salen) complexes into streptavidin. Their activities were improved by genetic optimization of protein scaffold and the ArM variants catalyzed the aziridination of styrene and oxidation of benzylic C−H bonds with up to 19 and 146 turnover numbers.

Abstract

The development of artificial metalloenzymes (ArMs) offers a potent approach to incorporate non-natural chemical reactions into biocatalysis. Here we report the assembly of Mn(salen)-based ArMs by embedding biotinylated Mn(salen) complexes into streptavidin (Sav) variants. Using commercially available nitrene and oxo transfer reagents, these biohybrid catalysts catalyzed the aziridination of alkenes and oxidation of benzylic C−H bonds with up to 19 and 146 turnover numbers.

The generic incorporation of a thioxanthone-containing amino acid into a protein scaffold is described. The resulting artificial photoenzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields with excellent enantioselectivity.

Abstract

Genetically encodable photosensitizers allow the design of artificial photoenzymes to expand the scope of abiological reactions. Herein, we report the genetic incorporation of a thioxanthone-containing amino acid into a protein scaffold via an engineered pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase pair. The designer enzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields (up to>99 % yield) with excellent enantioselectivity (up to 98 : 2 e.r.). This work provides a robust and facile method for photoenzyme design and lays the foundation for the development of further photoenzymatic reactions.

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 Communications, Published online: 28 November 2024; doi:10.1038/s41467-024-54613-3

There is a paucity of studies on enzyme stereoselectivity from an evolutionary biochemistry perspective. Here, the authors use ancestral sequence reconstruction to trace the evolution of stereoselectivity in imine reductases, elucidate its structural basis, and investigating the role of epistasis.

Science Advances, Volume 10, Issue 48, November 2024.

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.