Biocatalysis@TUDelft

Cyanobacterins are a class of bioactive natural products known for their potent algicidal and antitumor properties. In our previous work, we identified the cyanobacterin biosynthetic gene cluster and elucidated the biosynthesis of the furanolide core structure. However, the functions of all tailoring enzymes encoded within this cluster remained elusive. In this study, we utilized both, in vivo heterologous expression and in vitro biochemical assays, to investigate the roles of cyanobacterin biosynthetic tailoring enzymes. Extensive screening with a broad range of furanolide core structure analogs revealed a remarkable substrate promiscuity of these enzymes, making them valuable tools for biocatalytic late-stage diversification. In addition, an in-depth understanding of the biosynthetic sequence of the tailoring enzymes was obtained using early biosynthetic precursors: CybK, CybJ, and CybI sequentially orchestrate key cyanobacterin maturation steps, namely C10-hydroxylation, C10-O-methylation, and C8-chlorination, respectively. Following these modifications and subsequent furanolide core-structure assembly catalyzed by CybE/F, CybD installs the C17-methoxy group. Additionally, we identified and functionally characterized the ene-reductase CybG, which catalyzes the reduction of the C4-C13 double bond in cyanobacterins, transforming them into non-toxic saturated congeners. These insights not only enhance our understanding of the complex enzymatic pathway involved in cyanobacterin biosynthesis, but also facilitate the development of new tools for the enzymatic total synthesis of cyanobacterins and unnatural analogs, paving the way for future biotechnological applications.

Polyamides are important natural and synthetic polymers, best exemplified by proteins and nylons respectively. Proteins demonstrate that novel polymers with emergent properties can be generated by combining diverse monomers in precisely defined sequences. However, commercial polyamides represent only a small fraction of the potential diversity in polyamide sequences, due to the synthetic challenges of sequence-controlled polymerization. Amide synthetases have been shown to synthesize a broad array of nylon-relevant diads, but the generation of novel sequenced copolyamides requires enzymes capable of acting with longer and more diverse substrates. In this study, we demonstrated that NRPS-independent siderophore (NIS) synthetases, represented by DesD, can ligate oligomeric substrates. A simultaneous enzyme cascade using DesD enabled the synthesis of an oligotriad directly from unprotected substrates. Moreover, the regioselectivity of DesD allowed the selective synthesis of a sequenced amide tetrad, the precursor to a novel sequenced-defined polyamide with properties superior to nylon 66. This study establishes a direct biocatalytic route for the facile synthesis of sequenced oligoamides from unprotected bifunctional substrates, opening new possibilities to synthesize sequenced copolyamides.

Genetically encoded miniature photoenzyme miniSOG (12 kDa) enables spatiotemporally controlled bioorthogonal deboronative hydroxylation of 27 diverse organoboronates in live cells via localized superoxide radical anion O₂ ^•− generation.

Abstract

Artificial photoenzymes hold transformative potential for in vitro biocatalysis, but their translation to live-cell environments demands minimal cellular perturbation and aerobic compatibility. Here, we present miniSOG, a 12 kDa miniature photoenzyme that enables bioorthogonal deboronative hydroxylation via superoxide radical anion (O₂ ^•−) generation under blue light irradiation. Leveraging the inherent photochemistry of flavins, miniSOG facilitates the photoactivation of 27 structurally diverse organoboronates—including aryl/alkyl boronates, fluorophores, anticancer agents, and epigenetic modulators—through a unified O₂ ^•−-mediated mechanism. This system achieves spatiotemporally precise photocatalysis in live cells, where miniSOG's compact size and subcellular targeting enable organelle-specific localization and confined reactivity due to short-range O₂ ^•− diffusion (∼0.2 µm). We demonstrate its utility in light-gated cellular modulation: i) mitochondrial depolarization via localized release of 2,4-dinitrophenol (DNP) to disrupt energy metabolism, and ii) nuclear m⁶A methylation enhancement to epigenetically upregulate autophagy. By repurposing miniSOG's photochemistry for bioorthogonal deboronative hydroxylation, this work establishes a versatile, genetically encoded platform for manipulating fundamental cellular pathways with minimal off-target effects.

Proceedings of the National Academy of Sciences, Volume 122, Issue 44, November 2025.
SignificanceGlycoside hydrolases (GHs) perform diverse functions in organisms, with over 180 known enzyme families. Although hydrogen bonds and proton transfer play important roles in enzymatic reactions, hydrogen atoms are generally invisible in ...

Nature Chemistry, Published online: 04 November 2025; doi:10.1038/s41557-025-01979-6

Non-ribosomal peptide cyclases that catalyse head-to-tail macrocyclization are repurposed here to enable regioselective macrocyclization of branched peptides with multiple nucleophiles, affording lariat-shaped peptides with various sequences and ring sizes. Coupled with subsequent site-selective acylation chemistry, this chemoenzymatic approach facilitates modular access to structurally diverse lariat lipopeptides.

Cyclobutanone oxime was found to be efficiently converted to γ-sulfinylated nitrile by cleavage of the N─O and C─C bonds, followed by the C─S bond formation in aldoxime dehydratase, although only aldehyde oxime serves as a substrate in nature. This art personifies the function of the enzyme's active sites as carpenters specializing in shrine architecture. Details of the study are reported by Takashi Hayashi et al. in their Communication (e202511590).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) rely on a diverse array of enzymes to tailor peptide backbones and side chains. In this study, we characterized enzymes from two different biosynthetic gene clusters (BGCs) from Pseudomonas strains (pfl and pos) that catalyze new transformations in RiPP biosynthesis. Two -ketoglutarate-dependent HEXXH enzymes, PflC and PosC, perform hydroxylation of multiple consecutive glutamine residues and selectively recognize a C-terminal ARMD tetrapeptide to trigger oxidative backbone cleavage that generates an amide terminus. Mutational analysis pinpoints the first position of this motif as a critical determinant. Notably, PflC displays proteolytic activity in the absence of the leader peptide, indicating that leader peptide-enzyme interactions modulate the observed reaction selectivity. The biosynthetic gene clusters also encode a unique MNIO-nitroreductase fusion enzyme that installs a rare Z-dehydrophenylalanine and hydroxylates an Asp residue. Collectively, this work expands both the catalytic repertoire and structural diversity accessible through bacterial RiPP biosynthesis.

Massive DNA sequencing datasets are providing an unprecedented thesaurus of protein sequences. Still, when sequence homology to known enzymes is the only guide, their functional assignment and delineation of their catalytic strategies and mechanisms are lagging. The incremental nature of homology exploration is overcome by ultrahigh-throughput functional genomics, where jumps into unknown sequence space provide annotations without precedent. As a case in point, recent work has identified novel metal-free phosphotriesterases with a cysteine-containing triad in the active site capable of rate accelerations of up to 1013 in kcat/KM. Here we expand this exploration and observe sequence-structure-function relationships of a range of homologous proteins from the dienelactone hydrolase (DLH) family, revealing 10 new phosphotriesterases. Four new crystal structures provide clues to mechanism, suggesting - based on phylogenetic and structural analysis - that phosphotriesterase activity is mediated by lid loops surrounding the active site with activity correlated over 4 orders of magnitude to loop flexibility. These insights allow protein engineering by loop grafting across homologues, resulting in increased phosphotriesterase activity in a human enzyme. This exploration provides an annotation of starting points as well as an engineering strategy for the development of new reagents for bioremediation or treatment of organophosphate poisoning.

Nature Catalysis, Published online: 31 October 2025; doi:10.1038/s41929-025-01434-2

Constructing C(sp3)–C(sp3) bonds using non-prefunctionalized substrates as radical precursors is challenging. Now an ene-reductase and an organophotoredox catalyst work together to enable the enantiodivergent hydroalkylation of electron-deficient C(sp3)–H bonds via radical intermediates generated from carbanions.