Biocatalysis@TUDelft

Nosiheptide (NOS) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits potent antibiotic activity against multiple bacterial pathogens. NOS features a core macrocyclic peptide containing thiazoles, dehydrated serine and threonine residues, and a 3-hydroxypyridine ring. In addition to the macrocycle, NOS possesses a side-ring system formed by a 3-methyl-2-indolic acid (MIA) bridge that connects to glutamyl and cysteinyl residues on the core peptide via ester and thioester linkages. This unique side-ring is installed by the class C radical S-adenosylmethionine (SAM) methylase NosN. Here, we report X-ray crystal structures of the NosN homolog NocN--the first structure of a class C radical SAM methylase. The structures reveal clear electron density for two bound SAM molecules. Remarkably, the C5' atom of SAMI, which coordinates to the [Fe4S4] cluster, lies 3.1 [A] from the methyl group of SAMII and is properly positioned for direct hydrogen atom abstraction. A structure containing a product mimic illustrates how NocN engages its substrate and identifies Tyr276 as a key catalytic residue. The structure further suggests that the sulfonium center of SAMII may undergo epimerization to facilitate radical attack. Finally, electron paramagnetic resonance spectroscopy identifies a paramagnetic species consistent with the addition of the SAMII-derived methylene radical to the MIA substrate.

Bacterial aryl malonate decarboxylase is a cofactor-free enzyme that generates a wide spectrum of -chiral carboxylic acids in outstanding optical purity, including several non-steroidal anti-inflammatory drugs and chiral building blocks. The well-characterized AMDase from Bordetella bronchiseptica (BbAMDase) and related enzymes of the same family have three main limitations: (i) low stability, both operational and thermal, and (ii) limited substrate spectrum regarding the size of the smaller substituent on the -C-atom and (iii) low stereoselectivity towards -alkenyl--alkyl malonic acids. To address these limitations, we expanded the structural diversity of the AMDase family by ancestral sequence reconstruction (ASR). The phylogenetic analysis of the decarboxylase revealed conserved structural motifs and key amino acids in the hydrophobic active-site cavity, a catalytic motif crucial for activity and selectivity of the enzyme. The analysis highlighted the natural distribution of amino acid exchanges that had been previously identified in enzyme engineering campaigns. AMDase ancestors showed higher stability, activity, and, in one case, also stereoselectivity than BbAMDase. While the up to 10 {degrees}C higher unfolding temperature of AMDase ancestors is a frequent result in ASR, the improvement of the half-life time of 294-fold of ancestor N131 was surprising. Ancestor N31 formed 2-methyl-but-3-enoic acid from its corresponding malonic acid in an optical purity of 99.7% eeR. The extant BbAMDase produces this compound in much lower optical purity (96.8% eeR), which corresponds to a 1.4 kcal{middle dot}mol-1 difference of the transition state free energy of the two reaction paths leading to the different enantiomers. Furthermore, the stereoselectivity of the ancestors was completely inverted by switch of the catalytic cysteine residues G74C/C188G.

In its natural reaction, the fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) catalyzes the light-driven, redox-neutral decarboxylation of fatty acids via carbon-centered radical intermediates. Here, we systematically repurpose this radical pathway to unlock new reactivities, establishing CvFAP as a versatile platform for photobiocatalytic radical transformations. We expand its photocatalytic reaction portfolio by four new reactions: (i) the intermolecular Giese-type coupling of fatty acids with cycloalkenones, (ii) the intramolecular decarboxylative radical cyclization via nucleophilic radicals and isolated C=C bonds of the same philicity, (iii) a cysteine-mediated (Z)→(E) photoisomerization of unsaturated fatty acids, and (iv) a radical carbohydroxylation reaction that demonstrates the enzyme’s capability of olefin bis-functionalization. Enzyme Engineering, using computational modelling as basis, enhanced cyclization efficiency, demonstrating the evolvability of CvFAP towards new reactivities. The study expands the enzymatic repertoire of radical chemistry and demonstrates how photobiocatalysis can channel reactive intermediates to selective, new-to-nature bond-forming transformations, and pave the way toward enzyme-controlled radical couplings using abundant carboxylic acid feedstocks.

Membrane-bound alcohol dehydrogenase (ADH) from Gluconobacter oxydans is a direct electron transfer-type biocatalyst for ethanol oxidation. To improve its bioelectrocatalysis, the membrane-binding regions of ADH were predicted and then deleted. A soluble ADH variant without surfactants was purified from the soluble fraction and characterized using structural and bioelectrochemical approaches.

ABSTRACT

The design of artificial enzymes with non-canonical amino acids (ncAAs) capable of catalyzing non-natural reactions represents a promising frontier in biocatalysis. However, the synthetic challenges and high cost associated with ncAAs bearing catalytic residues have limited progress in this area. Here, we report the development of artificial Friedel–Crafts alkylases using a one-pot strategy that seamlessly integrates the biosynthesis of ncAAs with their site-specific incorporation into proteins via genetic code expansion. This innovative approach enables the functionalization of enzymes with novel catalytic properties tailored for stereoselective Friedel–Crafts alkylation. The artificial Friedel–Crafts alkylase exhibited efficient catalytic activity for the enantioselective Friedel–Crafts alkylation of enals and indoles via iminium activation, yielding a range of chiral indole alcohols with up to 98% enantiomeric excess (e.e.) and 99% yield following directed evolution. By demonstrating the feasibility and advantages of this one-pot strategy, we aim to establish a versatile platform for the design of artificial enzymes and to pave the way for broader applications in enzyme engineering and synthetic biology with in situ biosynthesized ncAAs.

Thiamine diphosphate (ThDP), or cocarboxylase, is successfully used as an N-heterocyclic carbene ligand for synthesis of a monodentate gold (I) complex, which efficiently catalyzes intramolecular hydroamination, hydroalkoxylation and hydrocarboxylation of a diverse array of alkyne substrates in water and open air at mild temperature.

ABSTRACT

Thiamine diphosphate (ThDP), or cocarboxylase, is an enzyme cofactor that catalyzes crucial metabolic reactions via a thiazolium carbene in all living systems. In this study, we successfully exploit this N-heterocyclic carbene ligand for the efficient synthesis of a monodentate gold (I) complex. The resulting ThDP = AuCl complex efficiently catalyzes intramolecular hydroamination, hydroalkoxylation, and hydrocarboxylation of a diverse array of alkyne substrates in water and open air at mild temperatures. This new metal catalyst is environmentally friendly and holds promise for the construction of novel artificial metalloenzymes.

The asymmetric synthesis of chiral sulfoxides remains challenging due to the lack of efficient screening methods. We developed a high-throughput colorimetric assay based on stereocomplementary sulfoxide reductases (MsrA/B), enabling simultaneous evaluation of activity and enantioselectivity. This platform successfully identified engineered unspecific peroxygenase variants with complementary stereopreference and offers a generalizable approach for developing stereoselective sulfoxidizing enzymes.

ABSTRACT

Chiral sulfoxides are important motifs in pharmaceuticals, yet their asymmetric synthesis remains challenging due to the need for stereocontrol. While bio-oxidation provides an attractive route, the lack of robust high-throughput screening methods hinders the development of enantioselective sulfoxide synthases. Herein, we developed a high-throughput assay combining sulfoxide reductases (MsrA/B) with a DL-dithiothreitol and 5,5’-dithiobis (2-nitrobenzoic acid) chromogenic system. This platform enables the simultaneous assessment of both catalytic efficiency and enantioselectivity, with colorimetric signals showing a linear correlation (R² = 0.99) against HPLC-validated enantiomeric excess. As a proof of concept, we screened 2880 engineered variants of recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO), a known thioether-oxidizing enzyme. This method allows the identification of mutants with complementary enantioselectivity (95.2% ee (R) or 10.3% ee (S)). In addition, the modular design of this assay—exploiting the stereo-complementarity of MsrA/B toward sulfoxides—provides a general framework for developing other stereoselective oxidoreductases for thioether oxidation.

Bump-and-hole studies on the Pseudomonas savastanoi ethylene-forming enzyme (PsEFE) enable formation of low levels of alkene products other than ethylene from naturally occurring 2-oxoglutarate derivatives. The results show that plants and/or microorganisms have potential for enzyme-catalyzed production of propylene and 1-butylene, potentially to exert distinctive signaling effects.

ABSTRACT

Ethylene is an established signaling molecule in plants and other organisms; however, the biosynthesis and biological roles of gaseous alkenes other than ethylene are less well defined. The Pseudomonas savastanoi ethylene/succinate-forming enzyme (PsEFE) catalyzes ethylene production from 2-oxoglutarate (2OG), though does not catalyze formation of alkenes from C4 alkyl- or hydroxyl-substituted 2OG derivatives. Here we report studies on the reactivity of L206 PsEFE variants with C4-substituted 2OG derivatives. Spectroscopic evidence reveals that L206V and L206A PsEFE react with C4-substituted 2OG derivatives to give diacid and alcohol products, similarly to wildtype (wt) PsEFE. Importantly, L206V PsEFE, but not L206A and L206G PsEFE, catalyzed production of low levels of acetaldehyde and propylene from the natural metabolites 4-hydroxy-2OG and 4-methyl-2OG, respectively. By contrast, L206A PsEFE, but not L206V and L206G PsEFE, catalyzed formation of low levels of 1-butylene from 4-ethyl-2OG. Together with studies from others, the combined results indicate the potential of bump-and-hole studies to modify the substrate and product selectivities of PsEFE reactions, provided that the PsEFE variant:2OG derivative pairs are matched. The results suggest that wildtype 2OG oxygenases other than PsEFE may catalyze production of gaseous alkenes other than ethylene.

Applied and Environmental Microbiology, Volume 92, Issue 2, February 2026.

Nature Chemical Biology, Published online: 19 January 2026; doi:10.1038/s41589-025-02129-2

Shin et al. report the cryo-electron microscopy structure and catalytic mechanism of TrhO, a cofactor-independent enzyme that hydroxylates tRNA wobble uridine using molecular oxygen through a reactive cysteine intermediate.

To overcome the low stereoselectivity of L-threonine aldolase (L-TA), a continuous-flow modular reactor with a chiral microenvironment is constructed. Its first module (L-TA@L-Arg-ZIF-67) enhances C_β stereoselectivity for converting 3,4-dihydroxybenzaldehyde and glycine to L-droxidopa, while the second module (YD@Ca₂(PO₄)₃) matches the catalytic rate required for norepinephrine (NE) biosynthesis. This integrated system boosts stereoselectivity and yield, enabling efficient NE production.

Abstract

Chirality lies at the core of pharmaceutical molecular design. Multi-enzyme cascade catalysis offers high stereoselectivity for constructing such molecules. Here, we present a continuous-flow modular reactor engineered with a chiral microenvironment for the biocatalytic synthesis of norepinephrine. This system incorporates two functional modules: a chiral catalytic reactor, designed to enhance the C_β stereoselectivity of L-threonine aldolase (L-TA), and a decarboxylation reactor to balance catalytic rates. Modifying the microenvironment surrounding L-TA significantly enhanced its stereoselectivity, with 4.1 times increased the de value (from 18.4% to 75.01%). Seen from molecular dynamics simulations, these modifications reshaped the spatial conformation of the enzyme's active site. Moreover, at an optimal column height ratio of 3:5 between the chiral and decarboxylation modules, norepinephrine yield reached 3.07 g/L. This modular reactor strategy, enhanced by a tailored chiral microenvironment, offers substantial promise for the stereoselective synthesis of pharmaceutical intermediates.

Characterization of anhydromevalonate phosphate decarboxylase, the UbiD-family decarboxylase involved in the archaeal mevalonate pathway, was conducted. The enzyme is responsible for the biosynthesis of isoprenoids, such as archaeal membrane lipids, respiratory quinones, and dolichols. Unexpected formation of byproducts during the reaction of the enzyme was shown to occur probably due to the vulnerability of the 1,3-dipolar cycloaddition adduct intermediate between the substrate trans-anhydromevalonate phosphate (tAHMP) and the cofactor prenylated FMN.

In the archaeal mevalonate pathway, the prototype of all existing mevalonate pathways, a unique intermediate, trans-anhydromevalonate phosphate, is decarboxylated to form isopentenyl phosphate. The key reaction is catalyzed by a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD) family decarboxylase, anhydromevalonate phosphate decarboxylase (EC:4.1.1.126). The yet-to-be-identified properties of the archaea-specific enzyme, such as the requirement for prenylated flavin mononucleotide (prFMN) as a coenzyme, were elucidated using an enzyme derived from the hyperthermophilic archaeon Aeropyrum pernix. The coenzyme can be supplied to the decarboxylase from coexisting prFMN synthase, which anaerobically catalyzes the prenylation of reduced flavin mononucleotide and subsequent cyclization. Kinetic analysis of A. pernix anhydromevalonate phosphate decarboxylase supported its physiological role in catalyzing the decarboxylation step and progressing the archaeal mevalonate pathway, which is characterized by lower ATP consumption than other mevalonate pathways and is therefore considered promising for future metabolic engineering. However, nuclear magnetic resonance and liquid chromatography–mass spectrometry analyses showed that the enzyme could form non-negligible amounts of secondary products, probably because of the reactivity of the intermediate cycloaddition adduct between prFMN and the substrate. This study provides deeper insights into the reaction mechanism of UbiD family decarboxylases via 1,3-dipolar cycloaddition.

Diols serve as important platform molecules, and their selective oxidation to high-value-added products holds significant importance. Herein, we show that the unspecific peroxygenases, upon immobilization, exhibit excellent enzyme activity in neat reaction conditions. This unique reaction systems allows selective conversion of diols to hemiacetals, and addresses the instability issues of hemiacetal molecules in aqueous phase environment.

Diols are versatile starting materials for preparing a series of value-added products. However, the biocatalytic oxidation of diols to hemiacetals remains challenging, primarily due to the inherent instability of hemiacetals in aqueous media. Herein, we report the use of unspecific peroxygenases for the mild and selective oxidation of diols into hemiacetal products. Based on the concept of reaction engineering, this catalytic process was performed under neat reaction condition using immobilized enzymes. This unique reaction system allowed a variety of patterned diols being converted into the stable hemiacetal products with chemoselectivity up to 99%. By optimizing the reaction conditions, the hemiacetal products were converted in situ to lactones, thereby further broadening the application of diol oxidation reactions. The molecular modeling of the enzyme–substrate interaction sets up a basis for the mechanistic understanding of the reaction activity and selectivity. This work demonstrated a new approach of transforming diols into synthetic building blocks by unspecific peroxygenases.

The structure of the non-canonical C₁₆ sesquiterpene named hegelenether was revised to the structure of marxdiol, and its absolute configuration was assigned. Isotopic labeling experiments revealed the stereochemical course of the methyltransferase involved in its biosynthesis. The enzyme mechanism was further addressed through protein X-ray crystallography and structure-based site-directed mutagenesis.

Abstract

Non-canonical methylation events generate terpene structures that evade classical biosynthetic predictions, as exemplified by the proposed C₁₆ terpene hegelenether. Here, we show that this natural product is misassigned and revise its structure to the dihydroxylated sesquiterpenoid marxdiol. Its absolute configuration and that of its precursor prekantenol pyrophosphate were determined through terpene synthase-mediated incorporation of stereoselectively labeled probes. To explain the initiating C6 methylation, we solved the crystal structure of the methyltransferase C6-FPP-MT with SAH and FPP, revealing a compact aromatic pocket that enforces Si-face methylation and Glu165-mediated deprotonation. These insights define how the active site controls regio- and stereochemistry and provide a structural basis for identifying related methyl-modified terpenes in uncharacterized biosynthetic pathways.

Rational protein engineering endows peroxygenases with high expression, improved activity and selectivity, and increased tolerance to H₂O₂, enabling efficient oxyfunctionalization of diverse substrates using hydrogen peroxide as a sustainable oxidant

Abstract

Selective C–H functionalization remains a central challenge in modern synthesis as it enables direct diversification of molecular scaffolds without pre-functionalization. Peroxygenases, including both unspecific peroxygenases and P450 peroxygenases, offer a biocatalytic solution to this challenge, catalyzing oxyfunctionalization reactions in aqueous media under mild conditions with hydrogen peroxide (H₂O₂) as the sole oxidant. In this review, we highlight recent advances in peroxygenase engineering over the past five years, with a particular focus on strategies that enhance heterologous expression, catalytic activity, and control of regio- and enantioselectivity. We also discuss protein engineering approaches that mitigate H₂O₂-induced inactivation and summarize efforts to repurpose NADPH-dependent P450 enzymes into self-sufficient peroxygenases, thereby expanding their catalytic repertoire. Furthermore, we examine the integration of peroxygenases with in situ H₂O₂ generation systems by enzymatic, chemical, photocatalytic, and electrochemical methods to achieve balanced oxidant delivery and sustained turnover. Collectively, these developments have established peroxygenases as versatile and robust catalysts for selective C–H functionalization, opening new opportunities for their application in the synthesis of pharmaceuticals, fine chemicals, and agrochemicals.

Proceedings of the National Academy of Sciences, Volume 123, Issue 2, January 2026.
SignificanceFlavodiiron proteins (FDPs) protect cells from nitric oxide (NO) toxicity by converting NO to harmless nitrous oxide (N2O) at a diiron active site. Despite extensive crystallographic and spectroscopic studies, the detailed structure and redox ...

ABSTRACT

Fungal unspecific peroxygenases (UPOs) have emerged as powerful catalysts for diverse oxidation reactions. While previous studies have predominantly focused on their native mono(per)oxygenase activity, their full catalytic potential remains underexplored. Herein, we demonstrate that the intrinsic peroxidative activity of UPOs can be effectively leveraged for the straightforward synthesis of benzoxazole compounds. Using the unspecific peroxygenase from Agrocybe aegerita, a broad range of ortho-patterned phenolic substrates were efficiently converted into high-value benzoxazoles with conversions of up to 99% under the neat reaction conditions. The enzyme exhibited superior catalytic performance compared to the well-established horseradish peroxidase. Mechanistic studies demonstrated that phenoxyl radicals generated by UPO's intrinsic peroxidase activity are essential for benzoxazole formation. This work not only presents a mild and efficient synthetic route to benzoxazoles but also expands the known reactivity and oxidative chemistry of UPOs.

Nature Synthesis, Published online: 13 January 2026; doi:10.1038/s44160-025-00972-8

In this Editorial, we reflect on some of the striking covers that Nature Synthesis published in 2025 and highlight the research they represent.