Biocatalysis@TUDelft

Nature Chemistry, Published online: 13 April 2026; doi:10.1038/s41557-026-02116-7

Hydrophobicity plays an important role in protein function, but tuning hydrophobicity with canonical amino acids is chemically limited. Now through the genetic incorporation of bulky, highly hydrophobic non-canonical amino acids, their utility in enzyme engineering by enhancing the function of a copper-dependent laccase through hydrophobic tuning has been demonstrated.

Nature Chemistry, Published online: 13 April 2026; doi:10.1038/s41557-026-02126-5

tRNA deacylases have evolved as resistance genes towards natural products that contain non-canonical amino acids by preventing their mistranslation. Now a strategy has been developed that leverages tRNA deacylases as class-agnostic genomic markers for amino acid-based biosynthetic gene clusters, identifying thousands of cryptic clusters and enabling the discovery of amino acid-based natural products.

Strictosidine synthases (STRs) are plant Pictet–Spenglerases catalyzing the coupling of a secoiridoid with tryptamine. All previously characterized strictosidine synthases are strictly S-selective. Here, we report the discovery of Epi-STR, an R-selective STR ortholog from the medicinal plant Pogonopus speciosus. We then engineer stereoselectivity in a canonical STR and Epi-STR through reciprocal amino acid substitutions.

ABSTRACT

The class-defining monoterpenoid indole alkaloid (MIA) scaffold strictosidine is generated by condensation of tryptamine and secologanin by the Pictet–Spenglerase strictosidine synthase (STR). All previously characterized STR orthologs are strictly 3S-stereoselective. Here, we report that the medicinal plant species Pogonopus speciosus (Rubiaceae) accumulates the 3R epimer vincosidic acid produced by a an ortholog of STR. This ortholog, named here Epi-STR, exclusively produces the 3R configuration, and is capable of accepting both secologanic acid and the methylester secologanin as aldehyde substrates. Using comparative phylogenetic and structural analyses, we determine the amino acid residues that confer stereoselectivity. Through rational design of reciprocal amino acid substitutions, we achieved switches in stereoselectivity in a canonical STR and in Epi-STR. The stereoselectivity of the engineered mutants is also dependent on the identity of the aldehyde substrate. Notably, previous and extensive engineering efforts have never been shown to switch the stereo-selectivity of STR. Therefore, this discovery now allows cost-effective epimer-pure access to the R-epimer, and offers mechanistic insights into the enzyme stereoselectivity of this important reaction. This work also highlights the importance of phytochemical analyses of poorly described plant species.

The vanadium-dependent haloperoxidase (VHPO) class of enzymes has been shown to catalyze cryptic nitrile oxide generation in an oxidative [3+2] cycloaddition reaction between aldoximes and alkenes to generate isoxazolines. This biotechnology has been applied to a chemoenzymatic sequence starting from aldehydes through in situ condensation with hydroxylamine and subsequent oxidative [3+2] cycloaddition.

ABSTRACT

Isoxazolines are an important class of heterocycles with a broad range of biological activities. One of the most prevalent synthetic strategies to access isoxazolines is through the oxidative [3+2] cycloaddition between nitrile oxides and alkenes initiated by halogenation of a starting aldoxime, but current methods rely on toxic oxidants that produce significant quantities of waste and are largely bioincompatible. We have recently discovered that the vanadium-dependent haloperoxidase (VHPO) class of enzymes are efficient catalysts for the in situ generation of nitrile oxides. Herein, we have developed a chemoenzymatic protocol for the conversion of aldehydes to nitrile oxides that features a sequential condensation of hydroxylamine with a starting aldehyde, followed by oxidative [3+2] cycloaddition with alkenes enabled by VHPO-catalyzed halogenation of aldoximes on a broad range of structurally diverse substrates in high yield and excellent chemoselectivity. The protocol is conducted on a gram scale, demonstrated using whole cells and cell lysate, and extended to isoxazole synthesis. Finally, this process is coupled to lipase-mediated conversion of amines to oximes to generate isoxazolines.

Use of the convertible nucleoside approach to synthesize m6Am RNA methyltransferase bisubstrates. This methodology allows for the introduction of modifications on the SAM analog moiety and the RNA substrate part, including the introduction of a cap analog by click chemistry. Docking studies performed on full-length methyltransferase PCIF1 generated by Alphafold 3 reveal interactions between the bisubstrates and the two substrate pockets.2

ABSTRACT

Phosphorylated CTD-interacting factor 1 (PCIF1) has been recently discovered to introduce a methyl group at the N6 position of the first transcribed Am-adenosine in nascent capped mRNA (m⁶Am modification), modulating mRNA stability, transcription, and translation. In addition, the activity of PCIF1 is involved in various diseases, making PCIF1 a potential therapeutic target. In the absence of complete crystal structures of PCIF1 with an RNA substrate and/or cofactor and with the aim of accessing potent inhibitors, the bisubstrate strategy was employed to synthesize a series of bisubstrate analogs of PCIF1 that contain an analog of the cofactor S-adenosyl-L-methionine (SAM) covalently linked to an Am-modified RNA substrate. The versatility of the synthesis was exploited to increase structural diversity at the amino acid chain of SAM, and a cap analog was introduced by click chemistry. Also, AlphaFold 3 was used to generate a complete structure of PCIF1 with an RNA substrate. Docking studies carried out with the bisubstrates indicate that the SAM analog occupies the cofactor binding site while the Am-RNA substrate interacts with the putative RNA pocket. We think that these results could be a valuable starting point for the design of potent inhibitors of PCIF1.

The Front Cover shows the 3D structure of the enzyme tryptophan synthase (TrpS) from Salmonella enterica serovar typhimurium, a versatile and efficient biocatalyst for the production of substituted L-tryptophans from indoles and L-serine. The already broad substrate scope of the enzyme has been further expanded in the Research Article by F. Parmeggiani and co-workers (DOI: 10.1002/cctc.202501808) to afford a range of non-natural tryptophans bearing substituents such as aryl rings, N-methylamino groups, alkynes, and halogens, as depicted in the background.

A Cupriavidus necator chassis couples a diamine oxidase–imine reductase cascade with an H₂-driven soluble hydrogenase cofactor module to convert linear diamines into saturated N-heterocycles in vivo, using H₂ as the sole source of reducing power.

ABSTRACT

Saturated N-heterocycles such as pyrrolidines and piperidines are core motifs in many agrochemicals and pharmaceutical agents, yet established chemical syntheses often rely on precious metals and face limitations in selectivity, scalability, and sustainability. Biocatalytic approaches offer high chemo- and stereoselectivity, but in vitro cascades require enzyme purification and external cofactor recycling, while whole-cell systems typically depend on carbohydrate feedstocks. Here, we report the engineering of the lithoautotrophic bacterium Cupriavidus necator for the hydrogen-driven conversion of linear diamines into saturated cyclic amines. Co-expression of diamine oxidases (DAOs) and an imine reductase (IRED) enabled an intracellular oxidation–reduction cascade powered exclusively by H₂. Notably, eukaryotic Cu/topaquinone-dependent DAOs were functionally produced alongside a bacterial oxidase, demonstrating efficient aerobic maturation of complex copper enzymes in a hydrogen-oxidizing host. Endogenous hydrogenases provide continuous NAD(P)H regeneration, decoupling reductive power from organic carbon metabolism. Process analysis identified gas–liquid mass transfer and redox balance as key determinants of productivity. This platform enables whole-cell synthesis of pyrrolidine- and piperidine-type products and represents, to our knowledge, the first H₂-powered whole-cell route from diamines to saturated N-heterocycles, providing a tunable blueprint for sustainable cyclic amine synthesis.

The aminoacylase MsAA is a suitable enzyme for the biotechnological production of N-acyl-L-amino acids. This study focuses on the optimization of the synthesis of N-lauroyl-L-methionine. A detailed analysis of the catalytic reaction mechanism of MsAA is performed in an in silico protein modeling study.

In this study, we present an investigation of the recently identified aminoacylase MsAA for the synthesis of N-acyl-L-amino acids, focusing on N-lauroyl-L-methionine. We found optimal reaction conditions at pH 8.0 and a temperature of 40–45°C with substrate concentrations of 400 mM methionine and 150 mM lauric acid. The highest product concentration of 100 mM was achieved with 67% substrate conversion after 72 h reaction at 40°C and pH 8.0. The reaction could be upscaled with a nearly identical reaction course. Several other fatty acids were also found to be substrates of this enzyme. Besides methionine, only hydrophobic amino acids were accepted for acylation. For a detailed analysis of the catalytic reaction mechanism of MsAA, we performed in silico protein modeling studies. Molecular docking of lauric acid and methionine to the predicted MsAA structure resulted in a similar mode of substrate binding as described for the related N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae. Differences in amino acid sequence of structurally conserved substrate-binding residues explain the distinct substrate scope of the enzymes. The structure–function relationship of relevant amino acid residues was validated by a mutagenesis study.

Fusing Aspergillus oryzae formate oxidase (AoFOx) to Curvularia inaequalis vanadium chloroperoxidase (CiVCPO) boosts soluble expression and enables formate-driven, in situ H₂O₂ supply for halogenation. Screening linker/orientation variants delivered up to 9-fold higher haloperoxidase activity in E. coli lysates, supporting arene bromination and bromolactonisation. Time courses and H₂O₂ spiking reveal hypobromite-induced AoFOx deactivation as the main robustness limit.

Vanadium–dependent haloperoxidases (VHPOs) are attractive biocatalysts for halofunctionalisation chemistry, but their routine use is frequently constrained by poor soluble recombinant expression. Here, we explore protein fusion as a construct-level strategy to simultaneously improve soluble expression of the vanadium chloroperoxidase from Curvularia inaequalis (CiVCPO) and enable in situ H₂O₂ generation via formate oxidase from Aspergillus oryzae (AoFOx). A panel of AoFOx–CiVCPO fusion designs was generated by varying enzyme orientation, linker length and linker architecture. Notably, fusion constructs displayed markedly increased haloperoxidase activity yields in crude lysates (up to ~9-fold relative to non-fused CiVCPO), whereas AoFOx activity decreased (approximately 36%–75%) compared to the individually expressed oxidase. A representative construct (CiVCPO–10 aa flexible linker–AoFOx) catalysed formate-driven bromination of activated arenes (phenol, thymol) and oxidative bromolactonisation of 4-pentenoic acid in crude extracts, giving product distributions consistent with hypobromite-mediated reactivity. Time-course experiments revealed that product formation was concentrated in the first 2 h and subsequently declined. H₂O₂-spiking partially restored activity, and sustained turnover was observed in a hypohalite-free sulfoxidation model reaction, implicating hypobromite-mediated deactivation of the AoFOx domain as a principal robustness-limiting factor.

Nature Biotechnology, Published online: 08 April 2026; doi:10.1038/s41587-026-03087-3

Sequence Display maps protein variant activities to a sequencing-based readout.

Strictosidine synthase (STR) is inactive toward N1-methyltryptamine due to steric clashes in the active site that raise reaction barriers. Computational insights guided the use of short-chain aliphatic aldehydes to relieve these clashes, enabling the asymmetric enzymatic synthesis of N9-methyl-tetrahydro-β-carboline derivatives.

ABSTRACT

Strictosidine synthase (STR) catalyzes in nature the enantioselective Pictet–Spengler condensation of secologanin and tryptamine to form (S)-strictosidine, a key tetrahydro-β-carboline intermediate in the monoterpene indole alkaloid biosynthesis. Extensive studies have revealed that STR exhibits a broad substrate scope, being capable of accepting short-chain aliphatic and aromatic aldehydes and tryptamine derivatives with substitutions at different carbon positions. However, the activity toward N1-substituted tryptamine derivatives remained unexplored. To address this gap, in the present study, molecular dynamics simulations and quantum mechanical calculations were performed to identify the reasons responsible for the previously reported inability of STR in catalyzing the reaction of 1-methyltryptamine with secologanin. It was revealed that this inactivity originates from kinetically prohibitive catalytic steps, caused mainly by the steric clashes in the active site introduced by the N1-methyl group, rather than substrate binding limitations. Guided by the structural insights, short-chain aliphatic aldehydes were predicted to alleviate these steric constraints, supported by calculated feasible reaction barriers. Experimental validation confirmed this prediction, enabling the successful asymmetric synthesis of multiple N9-methyl-tetrahydro-β-carboline derivatives. This work not only advances the fundamental understanding of STR catalysis but also establishes a combined computational-experimental strategy for exploring and extending the enzyme substrate scope.

Abstract/Summary ParagraphBile acids are steroidal metabolites produced by host and microbial metabolism that shape gut microbiome ecology and influence host physiology1,2. Bile acid structural diversification requires gut microbial bile salt hydrolase (BSH) activities, which cleave the amide bond of liver-derived bile acid amidates (BAAs)3. Beyond this gatekeeping metabolic function, BSHs expand the bile acid pool via their amine N-acyltransferase activity to produce numerous microbially derived BAAs that signal via host receptors and are further metabolized4,5. To date, all BSH activity has been attributed to a family of N-terminal nucleophile (Ntn) cysteine hydrolases. However, numerous gut anaerobic bacteria possess BSH activity but do not encode bsh genes from the Ntn family. Here, we describe a previously unknown class of metal-dependent BSHs (mBSHs) broadly distributed in the gut microbiome. These metalloenzymes have a distinct active site architecture from the canonical Ntn superfamily of cysteine hydolase BSHs (cBSHs) and are selective for taurine-conjugated BAAs. The discovery of this heretofore unappreciated class of BSHs overturns the paradigm that this important, conserved biochemical activity of the gut microbiota is provided exclusively by canonical cysteine hydrolases, greatly expands the known landscape of bile acid metabolism, and reveals a previously unrecognized link connecting host-microbiota bile acid co-metabolism with microbial taurine utilization pathways.

Nature Synthesis, Published online: 07 April 2026; doi:10.1038/s44160-026-01017-4

This Review explores biophotoelectrocatalysis, a biohybrid strategy that couples photo(electro)catalysts with enzymes and cells to drive selective chemical synthesis using sunlight. Recent advances, mechanistic insights and emerging applications—from photoenzymes to waste-to-value biorefineries—highlight the potential of biophotoelectrocatalysis for sustainable solar-to-chemical manufacturing.

Bile salt hydrolases (BSHs) are gut microbial enzymes that catalyze the deconjugation of glycine-or taurine-conjugated bile acids (BAs), a key step in shaping the BA pool in the human gastrointestinal tract and modulating host-gut microbiome interactions.1-3 All known BSHs are members of the N-terminal nucleophile (Ntn) hydrolase superfamily and share a conserved architecture and mechanism involving a nucleophilic active site cysteine.4,5 This knowledge has guided predictions and study of BSH activity in the gut microbiome6,7 as well as the development of BSH inhibitors8. Here, we report the discovery and characterization of a previously unknown BSH from the human gut bacterium Bilophila wadsworthia that belongs to the metal-dependent amidohydrolase superfamily and exhibits robust and specific activity toward taurine-conjugated bile salts. We show this secreted enzyme, metalloBSH, utilizes a metallocofactor for BA deconjugation, a mechanism distinct from that of canonical Ntn-type BSHs. MetalloBSHs are conserved in B. wadsworthia and present in many other Desulfovibrionaceae found in vertebrate gut microbiomes. Analysis of multi-omic datasets indicates metalloBSHs are expressed in vivo and correlate with BA metabolism. Overall, our findings reshape our understanding of BSH activity in the gut microbiome and highlight the promise of activity guided discovery in revealing previously overlooked gut microbial enzymes.

Polyphosphate kinases (PPKs) are revisited on their 70th anniversary, highlighting expanded nucleotide scope beyond ATP regeneration and emerging challenges in classification, stability, and phosphate accumulation. Clearer functional annotation and early integration of structural and process-level considerations are proposed to enhance rational engineering and broader application of PPK-based catalytic systems.

Polyphosphate kinases (PPKs) have long served as practical tools for ATP regeneration. Recent reports demonstrating broader nucleotide specificity have renewed interest in this enzyme family and expanded its perceived functional scope. At the same time, several recurring challenges have become more apparent, including ambiguity in classification, the frequent need for stability optimization, and the accumulation of phosphate by-products in coupled systems. These aspects do not represent insurmountable obstacles, but they influence how PPKs are compared, engineered, and integrated into catalytic platforms. This perspective argues that clearer functional annotation and early consideration of structural and process-level parameters can improve the interpretability and application potential of PPK-based systems.

Laccase-Catalyzed Dimerization: An exploration of fungal laccases as sustainable biocatalysts for the synthesis of bioactive neolignan dimers of honokiol and magnolol. The optimization of the dimerization conditions reveals an efficient approach to obtain natural and new dimers, whose improved in vitro inhibitory activity against metabolic enzymes highlights their potential application for treating metabolic diseases.

Laccases are biocatalysts with a high potential for developing green procedures to achieve new compounds. This study employed laccases in buffer solution to synthesize dimeric neolignans from honokiol and magnolol. Four laccases (LTV, POXA1b, EV3, and EV4) were first screened for honokiol dimerization in the presence of the mediator, with the variant EV4 achieving with the highest yield and in a shorter timeframe the natural houpulin A and B. Reaction conditions were optimized using a mathematical model to maximize the recovery of the desired products, achieving up to 3.42% yield. Interestingly, the optimized conditions also afforded the synthesis of other dimeric compounds, a magnolol dimer and a mixed honokiol–magnolol dimer with comparable yields. In silico studies investigated the substrate's compatibility with the EV4 binding site, highlighting the noncovalent interactions that enhance the stability of the radical formation, thus supporting the production of the obtained products. The four dimeric compounds were studied as metabolic enzyme inhibitors (lipase, α-amylase, and α-glucosidase) by in vitro and in silico experiments. The dimers exhibited more potent inhibitory activity than honokiol and magnolol, with houpulin B showing the strongest inhibition toward all tested enzymes.

Applied and Environmental Microbiology, Ahead of Print.

Carboxysomes are protein-based organelles that form the core of the bacterial CO2-concentrating mechanism (CCM) by elevating CO2 levels around Rubisco. They encapsulate Rubisco and carbonic anhydrase (CA) within a protein shell that, after closure, excludes cytosolic reductants. Because cytosolic CA activity would short-circuit the CCM, CA activity must be confined to the carboxysome, yet how -carboxysomes achieve this has remained unknown. Here we show that CsoSCA, the -carboxysomal CA, is redox-regulated: inactive under reducing conditions, active under oxidizing. This regulation is mediated by a conserved vicinal cysteine pair distal from the active site. CryoEM structures of Halothiobacillus neapolitanus CsoSCA under active and inactive conditions, and of an inactive cysteine variant, reveal that redox conditions modulate global conformational dynamics that reorganize the active site for catalysis. These findings advance the understanding of -carboxysome regulation and couple CsoSCA activation to lumenal oxidation during carboxysome maturation.