Biocatalysis@TUDelft

Using quantum mechanical/molecular mechanical calculations, the mechanism of the CC desaturation in anditomin biosynthesis is elucidated, catalyzed by the nonheme iron enzyme AndA. The results unveil a CO₂-mediated dual hydrogen atom transfer pathway, where CO₂ insertion suppresses hydroxyl rebound and generates a reactive Fe(III)-bicarbonate species to drive selective desaturation.

Fungal meroterpenoids, bioactive natural products with complex molecular frameworks, acquire their structural diversity partially through nonheme Fe(II)/α-ketoglutarate(αKG)-dependent enzymes. These enzymes use a high-valent Fe(IV)-oxo intermediate to drive diverse oxidative transformations. AndA, an Fe(II)/αKG oxygenase pivotal to anditomin biosynthesis, catalyzes regioselective C1C2 desaturation followed by skeletal rearrangement. While the isomerization step has been characterized, the mechanistic basis for desaturation over competing hydroxylation pathways remains enigmatic. Herein, molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations are employed to unravel how AndA avoids hydroxylation to achieve regioselective desaturation. The findings reveal the Fe(IV)-oxo intermediate, adopting two distinct coordination modes, a pentacoordinate (5C) and hexacoordinate (6C) geometry, differentiated by succinate coordination. The more reactive 5C species selectively abstracts the C2H hydrogen, initiating desaturation. Crucially, CO₂ generated in situ from αKG decarboxylation reacts with the resultant Fe(III)–OH complex, forming an Fe(III)-bicarbonate complex. This species sterically and electronically blocks OH rebound to the substrate. The Fe(III)-bicarbonate then abstracts a C1 hydrogen atom, completing the formation of the C1C2 double bond. These insights resolve the mechanism of AndA-catalyzed regioselective desaturation and demonstrate how CO₂-mediated coordination modulates oxidative fate, advancing mechanistic understanding of product control in this enzyme class.

Iron (II)- and 2-oxoglutarate-dependent (Fe(II)/2OG) oxygenases form a large family of non-heme enzymes containing the Fe(II) center coordinated by two histidine residues and either a carboxylate or halide ligand, with 2OG acting as a co-substrate. Although these enzymes share a conserved 2-His-1-carboxylate/halide motif in their active sites, they catalyze a wide variety of oxidative chemical reactions. We here investigate two factors that can significantly impact the divergence in their observed catalytic functions, namely, the intrinsic electric field (IEF) exerted on the active site by the surrounding protein environment, and variations of the composition of the facial triad. Concretely, we first evaluate the IEFs in Fe(II)/2OG oxygenases and investigate whether the direction and magnitude of these computed IEFs correlate with catalytic function across multiple subfamilies of Fe(II)/2OG oxygenases. We also examine how these IEFs can influence the geometric and electronic structures of Fe(III)-superoxo intermediates formed in the active site of Fe(II)/2OG oxygenases upon binding O2, the initial step of their oxidative catalytic cycles. Additionally, we evaluated how the identity and orientation of the third ligand (Glu, Asp, or Cl) in the 2-His-1-carboxylate/halide facial triad influence the active site complexes. Our findings suggest that specific steps in the catalytic cycle are determined by the interplay between the IEF due to the protein environment and the structural features of the facial triad. The results of this study provide insights into the role of IEFs and the facial triads in the observed reaction specificity of different Fe(II)/2OG enzymes.

Nature, Published online: 12 November 2025; doi:10.1038/s41586-025-09738-w

A new method for deracemization of atropisomers is described which leverages a P450 enzyme-mediated process involving bond rotation for enantioenrichment.

A sustainable one-pot/two-step photobiocatalytic cascade to convert α-methyl terminal alkenes into enantiomerically pure amines is developed. Visible-light-driven oxidative cleavage of alkenes to prochiral ketones using 9-fluorenone and molecular oxygen in dimethyl sulfoxide (DMSO) is seamlessly coupled with transaminase-catalyzed stereoselective reductive amination, affording pharmaceutically-relevant chiral amines in up to 95% overall conversion and >99% ee under mild and transition-metal-free conditions.

Merging photocatalysis with biocatalysis has recently emerged as a powerful strategy in chemical synthesis. Whereas photocatalysis enables the generation of reactive intermediates under mild conditions, biocatalysis provides exceptional selectivity with minimal byproducts. Herein, we report a one-pot/two-step photo-biocatalytic oxidation–reductive amination cascade that transforms α-methyl terminal alkenes into enantiomerically pure amines. In the initial step, 1,1-disubstituted (hetero)aryl alkyl olefins underwent oxidative cleavage to prochiral ketones using molecular oxygen under blue-light irradiation (427 nm) with 9-fluorenone as a transition-metal-free photocatalyst in dimethyl sulfoxide (DMSO), which simultaneously serves as solvent and quencher of in situ generated H₂O₂. Subsequent sequential combination with transaminase-catalyzed reductive amination furnished the targeted nonracemic amines in good-to-excellent overall conversions (52%–95%) and high-to-excellent enantiomeric excesses (90%–99.9%) in a stereocomplementary manner. The photo-biocatalytic cascade was successfully scaled up with α-methylstyrene (1.0 mmol), enabling the synthesis of the pharmaceutically relevant chiral precursor (R)-(+)-1-phenylethylamine in 93% conversion, 61% isolated yield, and >99% ee. Further derivatization of this key intermediate delivered the L-type calcium channel blocker fendiline in 77% isolated yield and 99% ee, without the need for costly transition metal catalysts, hazardous hydrogen gas, and high pressure.

Proceedings of the National Academy of Sciences, Volume 122, Issue 45, November 2025.

Proceedings of the National Academy of Sciences, Volume 122, Issue 45, November 2025.
SignificanceThe molecular mechanism by which metalloenzymes couple substrate binding and radical initiation is frequently enigmatic, even though such mechanisms are critical to achieving reaction specificity and preventing oxidative damage to itself or ...

QS-21, a saponin extract from the Chilean tree Quillaja saponaria, is gaining popularity as a potent vaccine adjuvant, but its production is constrained due to its low natural abundance and complex chemical structure of the triterpenoid saponins, which hinder large-scale production through plant extraction or chemical synthesis. Microbial biosynthesis presents a promising alternative, with Saccharomyces cerevisiae emerging as a desirable host for triterpenoid production. A key step toward microbial QS-21 synthesis is the efficient biosynthesis of its aglycone core, the triterpenoid quillaic acid. However, its efficient production remains limited by challenges of cytochrome P450 enzyme (CYP450s) activity, including cofactor availability and electron transfer efficiency. To address this limitation, we applied a multi-faceted metabolic engineering strategy to optimise CYP450 activity, including CYP450 expression and cytochrome P450 reductase (CPR) selection. Additionally, aligning CYP450 expression with the ethanol phase, enhanced the metabolic flux toward quillaic acid synthesis, leading to an 85-fold increase in titre. Together, these strategies led to a quillaic acid titre of 385 {+/-} 14 mg/L in flask fermentation. Fed-batch bioreactor fermentations increased quillaic acid titre to 471 {+/-} 20 mg/L and significantly increased the selectivity for QA from 32.6% to 65.1% of the total triterpenoids produced. These findings demonstrate the effectiveness of enhancing CYP450 activity through targeted strategies and reveal potential bottlenecks in CYP450 expression, providing valuable insights for future optimization of triterpenoid production in yeast.

Brewers yeast (Saccharomyces cerevisiae) acquires nitrogen from branched-chain and aromatic amino acids via the Ehrlich pathway, generating flavor (fusel) byproducts. Recently, diverting 4-hydroxyphenylacetaldehyde from Ehrlich catabolism of -tyrosine has enabled microbial production of opioids and other plant benzylisoquinolines. Yet, fusel metabolism is versatile in substrate scope, offering an untapped entry point for synthesizing structurally diverse aldehydes. Here, we repurpose the yeast Ehrlich pathway into a modular biocatalytic conduit for manufacturing privileged pharmaceutical alkaloids. We utilize retrobiosynthetic analysis and enzyme screening to derive scaffolds representative of solifenacin, colchicine, and ephedrine pharmaceuticals from simple amino acids. We survey wild yeasts for catabolism of -phenylglycine and demonstrate Ehrlich conversion to benzyl alcohol or (R)-phenylacetylcarbinol by 29 strains across nine genera. Implementing an {omega}-transaminase enables production of norephedrine from a simple amino acid input. This work unveils a generalizable biocatalytic route to clinically important alkaloids by exploiting metabolic logic from a yeast flavor pathway.

Unspecific peroxygenases (UPOs, EC. 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds due to their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have ever been reported in prokaryotic organisms. Here, we describe the discovery of a novel family of diverse bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial UPO-like proteins (BUPOs) are structurally analogous to family I ("short") fungal UPOs, despite having sequence similarity below 20%. Expression of one of these proteins (HydBUPO) in its native host (Hydrogenophaga sp. A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in Escherichia coli. In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, and two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol and 16-hydroxypalmitic acid, using hydrogen peroxide as co-substrate. These enzymes thus represent a new family of bacterial heme-thiolate peroxygenases that share structural and functional features with eukaryotic UPOs, offering new potential candidates for developing industrially relevant biocatalysts.

Lanmodulin-inspired Lanthanide-binding peptides synthesised in reversed order have a higher affinity to lanthanides than the natural sequence, as reported by Lena Daumann et al. in their Research Article (e202510453). As highlighted in this cover art the band of the cassette which gets rewound by the pencil symbolises the reversed peptides binding a trivalent europium. The original protein is shown as sketch in the back. That the higher affinity was a serendipitous finding is accentuated with the song title printed on the cassette.

Nature Chemical Biology, Published online: 11 November 2025; doi:10.1038/s41589-025-02061-5

Clostridium autoethanogenum produces ethanol from waste gases, but the biosynthetic pathway has been debated. Now, a combination of structural and biochemical data confirms that a key step in the ethanol biosynthesis pathway is acetate reduction by a tungsten-dependent aldehyde:ferredoxin oxido-reductase. This thermodynamically unfavorable reaction is counterbalanced by the coupling of ethanol synthesis with CO oxidation.

Science Advances, Volume 11, Issue 45, November 2025.

A nonheme iron enzyme, leucoanthocyanidin dioxygenase from Arabidopsis thaliana (AtLDOX), was repurposed to catalyze the enantioselective synthesis of a series of chiral α-amino acid derivatives via a 1,3-migratory nitrene C─H insertion process involving hydrogen atom transfer and radical rebound steps.

Abstract

Nonheme iron enzymes are among nature's most versatile catalysts for molecular functionalization. Engineering nonheme enzymes for abiological reactions unlocks new catalytic possibilities beyond the limits of natural evolution. In this work, we engineered a nonheme enzyme, leucoanthocyanidin dioxygenase from Arabidopsis thaliana (AtLDOX), to catalyze an asymmetric 1,3-migratory nitrene C(sp³)─H insertion reaction. Through directed evolution, the final optimized AtLDOX_LS variant efficiently delivers a range of chiral α-amino acids derivatives with exceptional activity and enantioselectivity (up to 81% yield, 850 total turnover number, and 98:2 enantiomeric ratio). Preliminary mechanistic studies suggest the involvement of radical intermediates for this transformation. This work advances the biocatalytic toolbox for radical involved transformations and broadens the scope of enzymatic migration chemistry.

Burkholderia pseudomallei and Burkholderia mallei are dangerous pathogens that cause severe diseases with high mortality rates. Their virulence relies in part on malleicyprols, potent toxins containing a highly reactive cyclopropanol group. In this study, we identify BurK, a heme-dependent oxidoreductase that neutralizes malleicyprols by enzymatically opening the cyclopropanol ring.

Abstract

Pathogenic bacteria of the Burkholderia pseudomallei group cause life-threatening infections in humans and animals. Their virulence factors include malleicyprols bearing a reactive cyclopropanol moiety essential for toxicity. Inactivating this reactive motif, therefore, is a promising way to neutralize these toxins. Here, we identify a heme-dependent oxidoreductase (BurK) that cleaves the cyclopropanol warhead. Mutational analyses and in vivo radical capturing show that BurK catalyzes a radical ring opening to yield a propanone fragment. Characterizing BurK orthologs across various bacterial phyla suggests broader ecological roles of these unusual enzymes. Using a nematode model, we demonstrate that BurK-producing helper bacteria neutralize malleicyprols, significantly reducing toxicity and enhancing host survival. In addition to uncovering a novel biocatalyst, this work lays the foundation for antivirulence approaches using therapeutic microbes against antibiotic-resistant pathogens.

Integrating a metallocofactor (Vitamin B₁₂) with an immobilized enzyme enables a recyclable chemoenzymatic cascade. Vitamin B₁₂ first catalyzes the deprotection of allyl ether to alcohol, which is subsequently converted by immobilized lipase into the target ester. The immobilized lipase boosts activity beyond free lipase and Novozym 435, and the cascade retains > 90% of its initial activity after five cycles.

Chemoenzymatic cascades combine the strengths of chemical and biological catalysis, offering tremendous potential for applications in synthetic chemistry, but integrating natural metallocofactors with immobilized enzymes in aqueous media remains underdeveloped. Here, we report the example of a recyclable chemoenzymatic cascade through integrating a natural metal-based catalyst (vitamin B₁₂) with an immobilized enzyme. By a two-step-sequence chemoenzymatic cascade, vitamin B₁₂ first catalyzes a deprotection of allyl ether to release 3-phenyl-1-propanol, which is subsequently converted by immobilized Candida antarctica lipase B (CalB) into the target ester 3-phenylpropyl butyrate. CalB is immobilized on tailored alkyl-functionalized silica nanoparticles, creating a hydrophobic microenvironment that enhances enzyme immobilization and good chemoenzymatic cascade activity, outperforming the controls (free enzyme and Novozyme 435). Under optimized conditions, the designed cascade reaction achieves 62% conversion in 24 h, and interestingly, the system can be reused for at least five cycles while retaining over 90% of its initial activity. As a compelling demonstration of integrating a natural metal complex with immobilized enzymes in a cascade process, this article establishes a good example for chemoenzymatic synthesis, with broad potential for extension to other high-value chemical transformations.

Nature Catalysis, Published online: 03 November 2025; doi:10.1038/s41929-025-01436-0

The creation of artificial metalloenzymes compatible with complex biological settings could enable broad applications. Now a de novo-designed artificial metalloenzyme containing an abiological ruthenium cofactor is reported and optimized for ring-closing metathesis in the cytoplasm of whole cells.

Nature Catalysis, Published online: 03 November 2025; doi:10.1038/s41929-025-01435-1

Radical repositioning to activate remote bonds is underdeveloped in synthetic biocatalysis. Now a photobiocatalytic system couples light-driven single-electron transfer and the relocation of unpaired electrons to activate remote C–C and C–H bonds for enzymatically controlled enantioselective acylation.