Biocatalysis@TUDelft

Quinolones are valuable scaffolds for drug discovery but are rare in nature. Here, we show that two fungal enzymes, AthePKS and FerePKS, can generate 2-quinolones and two additional heteroaromatic scaffolds. Using AthePKS, we designed an artificial enzymatic cascade towards an antimicrobial quinolone from a simple precursor and implemented it in E. coli. We also determined the AthePKS crystal structure, suggesting hotspots for improving its catalytic efficiency by protein engineering.

Quinolones are privileged scaffolds for drug discovery that are relatively rare in nature. Here, we characterise two promiscuous fungal polyketide synthases AthePKS and FerePKS, which we had previously found to produce 2-quinolones in vitro. We challenged the enzymes with several substituted anthranilic acid derivatives, revealing their ability to produce precursors of pharmaceutically relevant quinolones. We also discovered that AthePKS and FerePKS accept other 2-substituted benzoic acids, leading to the formation of coumarin and thiocoumarin scaffolds. We applied AthePKS in an artificial enzymatic cascade towards an antimicrobial 4-methoxy-1-methyl-2-quinolone and demonstrated its in vivo feasibility by successfully expressing the pathway in Escherichia coli. Lastly, we determined the crystal structure of AthePKS, suggesting hotspots for enhancing its catalytic efficiency by enzyme engineering. Our results provide a framework for further engineering of enzymatic routes towards privileged heteroaromatic scaffolds and derivatives thereof.

Nature Chemistry, Published online: 02 January 2026; doi:10.1038/s41557-025-02032-2

Catalysis has been a standard topic taught in university chemistry courses over the past century yet biocatalysis — or enzyme catalysis — has only recently been integrated into standard chemistry curriculum despite its broad applicability in industry. In a fourth year undergraduate research project course, students can now choose to explore interesting chemical transformations in the lab using biocatalysis instead of traditional synthetic chemistry approaches.

Conversion of emestrin J (5) to emestrin (1) by three P450 enzymes from the cluster (eme) in Emericella quadrilineata. EmeO acts as a bifunctional enzyme for the construction of the 15-membered lactone ring via an aryl-aryl ether bond formation and simultaneous hydroxylation between phenolic and nonphenolic aromatic rings, while EmeE and EmeR install regioselective and stereospecific hydroxyl groups at the ß-positions of the diketopiperazine ring.

ABSTRACT

Emestrins, a subgroup of epipolythiodioxopiperazines, are originated from cyclo-l-Phe-l-Phe and feature a dihydrooxepine ring. They contain typically a 15-membered lactone ring with an aryl-aryl ether linkage. Despite considerable progress in elucidating epipolythiodioxopiperazine biosynthesis, the enzymatic mechanism for the ether bond formation in emestrins remains uncharacterized. We identified a putative gene cluster (eme) in the fungus Emericella quadrilineata with three unknown P450 enzymes, EmeE, EmeR, and EmeO. Gene deletion, feeding experiments, and in vitro assays proved that EmeE and EmeR install regioselective and stereospecific hydroxyl groups at the ß-positions of the diketopiperazine ring. EmeO acts as a bifunctional enzyme for the construction of the lactone ring via an aryl-aryl ether bond formation and simultaneous hydroxylation between phenolic and nonphenolic aromatic rings. To the best of our knowledge, such enzymatic reactions have not been reported prior to this study.

One-step synthesis of complex alkyl oxystilbenin glucosides (6-47% yield) in alcoholic medium. Switching to ter-BuOH as solvent afforded δ-Viniferin diglucoside, with high selectivity and high yield (> 90%), that can be upgraded by enzymatic esterification to alkyl δ-Viniferin diglucosides. These new compounds exhibit tunable hydrophilic/lipophilic balance and valuable antioxidant and anti-inflammatory properties.

ABSTRACT

Stilbenes, such as resveratrol, possess numerous biological activities desirable in various industrial sectors (e.g., pharmaceutical, nutraceutical, and cosmetic). However, these activities are counteracted by their low bioavailability. In order to achieve an optimal hydrophylic/lipophilic balance (HLB) and thus maximize bioavailability, alkyl glucoside dimers of resveratrol (i.e., C2 to C12 alkyl oxystilbenin diglucosides (C8-O4’ linkage) and δ-Viniferin diglucoside (C3-C8’ linkage)) were synthesized from piceid—a β-glucoside of resveratrol—in one or two steps using a silver acetate-mediated oxidative coupling in various alcoholic solvents as the key step. δ-Viniferin diglucoside was functionalized by lauric acid using an enzyme-mediated esterification. HLB values, determined from Log P (the partition coefficient between water and octan-1-ol), were calculated for the nine synthesized compounds, revealing that four exhibited values within the range considered favorable for bioabsorption. Finally, antiradical and antioxidant activities were assessed using DPPH and ORAC tests, respectively, prior to anti-inflammatory testing on dermal human cells. These assays have shown very promising results with some activities overpassing that of resveratrol.

Organofunctional silanes are valuable in materials science as crosslinkers and adhesion promoters, but their synthesis typically requires costly transition-metal catalysts. We developed a sustainable biocatalytic platform for enantioselective synthesis of ester- and cyano-functionalized silanes. Directed evolution of Thermus amyloliquefaciens protoglobin (TamPgb) yielded enzymes that cyclopropanate vinyl silanes/siloxanes with diazo compounds or N-tosylhydrazones as carbene precursors (up to 2500 TTN, >99% dr/ee). This safer, practical method avoids precious metals and simplifies purification.

Abstract

Organofunctional silanes are highly useful reagents in material science, functioning as effective crosslinking agents and adhesion promoters. Traditionally, their synthesis relies on precious transition-metal catalysts, which require downstream purification and increase overall costs. Herein, we present a green and sustainable biocatalytic platform for the enantioselective synthesis of diverse organofunctional silanes bearing ester and cyano groups. Directed evolution of Thermus amyloliquefaciens protoglobin (TamPgb) produced efficient enzymes for cyclopropanation of vinyl silanes and siloxanes using diazo compounds and N-tosylhydrazones as carbene precursors with excellent diastereo- and enantiocontrol (up to 2500 TTN, >99% dr, >99% ee). Notably, we demonstrate for the first time that N-tosylhydrazones can serve as carbene precursors in enzymatic reactions, providing a safer and more practical alternative for industrial applications.

The image illustrates the temperature induced change in hydrogen bonding pattern (room temperature at top right; and 120°C at bottom left) for rubredoxin protein (in cartoon form) from hyperthermophile archaeon Pyrococcus furiosus (in orange) living at boiling temperatures at undersea vents. More in the Research Article (e20302), Stephen P. Cramer and co-workers. Cover art credit: Ewa Kijowska-Stroes.

Proceedings of the National Academy of Sciences, Volume 122, Issue 52, December 2025.
SignificanceThe human gut microbiome modulates the health effects of dietary compounds by modifying their chemical structures. Gut microbes extensively metabolize polyphenols, a group of diverse plant-derived compounds associated with positive health ...

Amino acids, peptides, and proteins play pivotal roles in medicine, materials, and catalysis, with their functions largely dictated by their side-chain functionality. Recent studies have shown that heme oxygenase-like domain-containing oxidases (HDOs) catalyze a broad range of interesting chemical reactions on amino acids and their derivatives to produce structurally diverse target structures. Using a bioinformatics approach, the mangotoxin biosynthetic operon was found to house an unannotated HDO, MboA, along with a dedicated redox partner, MboB. We show that MboA catalyzes alkyne formation by iterative desaturations on the side-chain of a peptide substrate, where the transformation between alkene and alkyne is gated by MboB. The crystal structure of Fe(II)2-MboA reveals an unexpectedly short Fe–Fe distance, suggesting that the activation of strong C(sp2)-H bonds may utilize a mechanism distinct from other HDOs and expands the scope of known HDO chemistry.

The past decade has witnessed groundbreaking developments in metalloenzyme-catalyzed free radical transformations which were previously unknown or uncommon in native metalloenzymology. Guided by mechanistic understandings from organic, organometallic and biochemistry, an array of radical reactions has been developed using various metalloprotein catalysts based on first-row transition metal cofactors including Fe, Co and Cu. The structural and functional diversity and the readily tunable active-site environment of metalloproteins offer an excellent opportunity to solve the challenging chemo-, regio- and stereoselectivity problems in radical-mediated transformations facing synthetic chemists. In this Review, we summarize metalloprotein-catalyzed radical reactions based on the reactive intermediates involved, including carbon-centered radicals, nitrogen-centered radicals, oxygen-centered radicals, and metal carbenoids and nitrenoids with radical character. We further survey the reaction mechanism, enzyme engineering strategies, and substrate scope of these metalloprotein-catalyzed radical transformations, providing an overview of the current status of metalloenzymology unknown or uncommon in native biochemistry.

The 21st century has witnessed rapid advancements in synthetic biology, with DNA synthesis emerging as a foundational technology. Conventional phosphoramidite-based methods face significant limitations, including short DNA elongation lengths (<300 nt), hazardous chemical waste, and low stepwise incorporation efficiency. Enzymatic DNA synthesis using terminal deoxynucleotidyl transferase (TdT) offers a promising alternative, enabling kilobase-scale assembly with greater efficiency and minimal environmental impact. Here, we identified Bos taurus TdT (BtTdT) through UniProt database mining as a catalytically active scaffold for natural and 3'-modified dNTPs. Comprehensive characterization of BtTdTs enzymatic properties--including pH, temperature, metal ion dependence, and substrate specificity--revealed its optimal conditions. Truncation of the BRCT domain generated variants with enhanced activity compared to wild-type BtTdT. Guided by AlphaFold3-predicted structural models, we engineered a quintuple mutant (M5: Bt15AAR336L/K338G/L397M/E456S/D395G) optimized for 3'-ONH2-dNTP incorporation. M5 exhibited 30-fold activity enhancement relative to the triple mutant M3 (Bt15AAR336L/K338G/L397M) and achieved stepwise incorporation efficiency exceeding 98% in de novo synthesis of 10-nt ssDNA, demonstrating its potential for scalable enzymatic DNA synthesis. This work establishes a rational framework for TdT engineering through rational domain truncation and computational design, showing potential toward industrial-scale enzymatic DNA manufacturing.

Heat capacity changes are increasingly recognised as crucial for understanding the temperature dependence of enzyme-catalysed reaction rates. Here, we combine experiments and molecular dynamics simulations to investigate triosephosphate isomerase (TIM, TPI) from psychrophilic, mesophilic and thermophilic organisms. Kinetic data show clear curvature in rate-temperature plots, particularly for the cold-adapted enzyme, independent of unfolding. This non-Arrhenius behaviour is accounted for by Macromolecular Rate Theory (MMRT), implying an activation heat capacity, largest for the psychrophile. Simulations reveal the molecular origins of these differences, showing significant conformational and dynamical changes between reactant and transition states in the cold-adapted enzyme, with a smaller activation heat capacity in the mesophilic enzyme, and near-zero for the thermophile. Transition-state-like conformations and reorganized correlated dynamics underlie these adaptations. These results demonstrate that dynamical differences between crucial states along the reaction pathway play a key role in determining the optimum temperature of activity for this archetypal enzyme.

Lenacapavir is a first-in-class viral capsid inhibitor, approved for the treatment of HIV, and which has been shown to give almost complete protection against transmission of the virus. However the high costs currently associated with the manufacture of Lenacapavir place restrictions on its wider application, making the development of cheaper synthetic routes a key priority. Herein we report the development of an engineered aminotransferase for the synthesis of the central chiral amine core of Lenacapavir. Due to the sterically demanding nature of the ketone substrate, a substrate walking approach was adopted during directed evolution to unlock desired aminotransferase activity starting from a parent template with no observable activity for the target reaction. Introduction of ten mutations into an aminotransferase from Ruegeria sp. TM1040 led to the development of a robust engineered aminotransferase that allowed production of the target chiral amine product with high conversions (>98%), isolated yields (>90%) and optical purity (>99% e.e.). This engineered enzyme will facilitate the development of a biocatalytic process to a key chiral intermediate of Lenacapavir in order to reduce its manufacturing costs and thereby enable global access to this important therapy.