Biocatalysis@TUDelft

Nature Biotechnology, Published online: 24 October 2025; doi:10.1038/s41587-025-02859-7

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In a recent study, Bachar et al. developed an enzymatic biocathode-based bioelectrocatalytic platform for efficient synthesis of chiral compounds. They then integrated this system with a semiconductor-based photoanode to construct a light-driven, bias-free photobioelectrochemical cell (PBEC). The system is versatile and adaptable for any process requiring NADPH-dependent enzymes, in vivo or in vitro.

Nature Catalysis, Published online: 24 October 2025; doi:10.1038/s41929-025-01433-3

Artificial photobiocatalytic reactions are appealing but sometimes suffer from non-enzymatic side reactions. Now a photoenzyme for enantioselective [2 + 2] photocycloaddition of 2-naphthyl derivatives is reported and combined with designed quenchers that shut down the competing enzyme-free racemic reaction.

Nature Catalysis, Published online: 23 October 2025; doi:10.1038/s41929-025-01426-2

Methylthio-alkane reductases are recently discovered enzymes that can produce methanethiol and small hydrocarbons from methylated sulfur compounds. Now the cryo-EM structure of a methylthio-alkane reductase complex is solved, revealing large metalloclusters previously observed only within nitrogenases.

Nature Chemistry, Published online: 21 October 2025; doi:10.1038/s41557-025-01958-x

Azetidine is a four-membered aza-cycle important in medicinal and organic chemistry. This study describes a mechanism of azetidine amino acid biosynthesis from l-isoleucine or l-valine by two non-haem Fe enzymes, PolF and PolE, in the polyoxin antifungal biosynthetic pathway.

Nature, Published online: 15 October 2025; doi:10.1038/s41586-025-09576-w

Bacterial ATP-binding cassette (ABC) transporters can be utilized and engineered to transport non-canonical amino acids into Escherichia coli for highly efficient synthesis of proteins with novel functions.

Nature Synthesis, Published online: 21 October 2025; doi:10.1038/s44160-025-00907-3

Rotenoids are natural insecticides from Fabaceae plants. Here the complete biosynthetic pathway to make rotenoids is elucidated in Amorpha fruticosa and Tephrosia vogelii, revealing a unique Fe(II)-dependent dioxygenase that catalyses core B-ring formation via redox-neutral cyclization. These findings enable the production of rotenoids in tobacco, paving the way for future biotechnological production.

TmT1βH, a novel taxane C1β-hydroxylase belonging to the α-KG/Fe(II)-dependent dioxygenase from Taxus × media cell cultures that catalyzes 1-dehydroxybaccatin IV (1) to form a major product baccatin IV (1a) and its isomer (1b). Tm576, a novel dedicated α-KG/Fe(II)-dependent dioxygenase that was able to convert 1 to 1b specifically. A mechanism that the 5/7/6-membered carbon framework arises from prototypical 6/8/6-type taxane skeleton via radical rearrangement was proposed.

Abstract

Here, we report the discovery and functional characterization of one novel taxane C1β-hydroxylase (TmT1βH), belonging to the α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenase family from Taxus × media cell cultures. The incubation of recombinant TmT1βH with 1β-dehydroxybaccatin IV (1) as a substrate led to the production of a major C1-hydroxylated product, baccatin IV (1a), and a minor product, 15-hydroxy-11(15→1)abeo-baccatin IV (1b), a non-classical 5/7/6-type taxane. Moreover, in vitro biochemical assays, molecular docking, and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for TmT1βH catalysis. Substrate specificity investigations revealed that TmT1βH preferred taxoids with high oxygenation level. Notably, we have also discovered a novel specific enzyme (Tm576) belonging to α-KG/Fe(II)-dependent dioxygenase that was able to convert 1 to 1b independently. A mechanism that the 5/7/6-membered carbon framework arises from prototypical 6/8/6-type taxane skeleton via radical rearrangement was proposed based on DFT calculations. More importantly, we artificially reconstructed the biosynthetic pathway of two important taxanes, baccatin IV, and baccatin VI, from GGPP in tobacco system. This work not only fully characterizes the role of C1β-hydroxylase of taxoids, but also offered new insights into the formation of taxane structural diversity.

Four novel cytochrome P450 enzymes, CsCYP1–CsCYP4, were functionally characterised for catalysing the formation of the characteristic 13,17-lactone, 5,19-lactone, and tropone moieties in the biosynthesis of cephalotane-type diterpenoids. Accordingly, the co-expression of these characterised CYP450 enzymes along with cephalotene synthase (CsCTS) in Nicotiana benthamiana enables the production of a wide range of cephalotane-type diterpenoids.

Abstract

Cephalotane-type diterpenoids, a class of natural products exclusively found in Cephalotaxus plants, are well known for their attractive structures and potent biological activities. However, their low natural abundance and intricate cage-like structures hinder their accessibility. Recently, the identification of a cephalotene synthase (CsCTS) has addressed the first committed step in the biosynthesis. However, the enzymes involved in the complex post-modification of the cephalotene core into structurally diverse cephalotane-type diterpenoids remain obscure. In this study, we functionally characterised four novel cytochrome P450 enzymes from C. sinensis. These enzymes demonstrate multiple oxidative functions and cooperatively catalyse a cascade of oxidation reactions, including the formation of signature 13,17-lactone, 5,19-lactone, and tropone. We further co-expressed the characterised CYP450 enzymes in combination with CsCTS to produce a variety of cephalotane-type diterpenoids, including hainanolidol (2), mannolide C (3), mannolide A (4), and cephinoid H (5), in Nicotiana benthamiana. Subsequently, harringtonolide (1) was chemically converted from hainanolidol (3.9 µmol with 10 equiv of Pb(OAc)₄) in 87.5% yield. In this study, the biosynthetic pathways of representative cephalotane-type diterpenoids were elucidated and reconstructed, thereby establishing a foundation for their sustainable production through biosynthesis and/or chemo-biosynthesis, highlighting the remarkable efficiency of merely five Cephalotaxus-specific enzymes in assembling such structurally complex natural products.

The bacterial diterpene synthase CjCS for chryseojoostenes was mechanistically characterised through labelling experiments, density functional theory (DFT) calculations, and conversion of substrate analogs with blocked reactivity. Site-directed mutagenesis gave access to minor enzyme products, including chryseojoostene E, whose biosynthesis involves a long-range hydrogen shift that is also relevant to its EIMS fragmentation.

Abstract

A diterpene synthase from Chryseobacterium joostei was characterised and produces the five unique compounds chryseojoostenes A–E. Chryseojoostenes D and E were produced in too low amounts for isolation from the wildtype enzyme, but extensive site-directed mutagenesis resulted in an enzyme variant in which the production of these compounds was enhanced. The biosynthesis of the enzyme products was investigated in detail through a combined experimental and computational approach, indicating a complex hydrogen scrambling during terpene cyclisation and a long-range proton shift towards chryseojoostene E. Density functional theory (DFT) calculations revealed that a similar long range hydrogen shift is involved in the formation of an even fragment ion (m/z 216), characterising the unique chemistry of the chryseojoostene skeleton. Further insights into the cyclisation mechanism were obtained by enzymatic conversion of two substrate analogs with reduced reactivity.

Enoyl-CoA carboxylases/reductases (ECRs) are enzymes with the fastest carbon dioxide (CO2) fixation capabilities, yet the precise mechanisms behind their assembly and catalytic activity are structurally not yet fully understood. Here, we employed cryo X-ray crystallography to reveal the dimeric structural organization of a novel ECR, isolated from mesophilic Mesorhizobium metallidurans (M. metallidurans). We examined the interactions in silico and compared oligomerization of our dimeric ECR from M. metallidurans (ECRMm_Dim) with tetrameric ECR from Burkholderia ambifaria (ECRBa_Tet)by using size exclusion chromatography in solution. Our in silico analysis revealed that specific residues in the M. metallidurans ECR that preclude tetramer formation, which could affect the enzymes catalytic activity. Additionally, we compared primary ECR sequences and structural variations between K. setae and M. metallidurans to explore their evolutionary relationships, along with their functional diversity. Our study presents the first example of a dimeric ECR structure which may provide new insights into how dimerization versus tetramerization may have an impact on catalytic function. By detailing how different oligomeric states influence enzyme activity and exploring active site conformational changes, we may offer a further understanding of ECR assembly. This work paves the way for future research into the precise molecular mechanisms that drive ECRs exceptional overall catalytic activity, efficiency and efficacy.

A dynamics-driven fragment-based approach reveals a cryptic pocket in EcDsbA, enabling the rational design of inhibitors beyond its canonical substrate-binding site.

Abstract

We have used nuclear magnetic resonance (NMR) spectroscopy to characterize dynamics in the bacterial oxidoreductase enzyme Escherichia coli disulfide bond protein A (EcDsbA). Through this process we identified a cryptic pocket in the structure. We demonstrate that we can identify small molecule “fragments” that bind entirely within this cryptic site. The fragments bind to the cryptic pocket with unusually slow kinetics and a preference for interacting with the oxidized state of EcDsbA where the two cysteine residues at the active site form a disulfide bond. We characterize the mechanism of binding, involving conformational changes in the active-site helix of EcDsbA, which are observed preferentially in the oxidized state. This dynamics-driven binding mechanism explains both the slow kinetics and the redox-dependent binding of the ligands. Furthermore, we demonstrate that compounds binding to the cryptic pocket inhibit EcDsbA activity. These findings highlight the value of dynamics data in identification of the cryptic pocket and identify a new target site for developing more potent inhibitors of EcDsbA.