Biocatalysis@TUDelft

Kaitocephalin (KCP) is a fungal neuroactive natural product bearing a peptide-like yet nonpeptidic amino acid-derived scaffold in which amino acid-like units are connected by C-C bonds rather than peptide bonds. The enzymatic construction of this unusual scaffold has remained unresolved. Here, we identify KpbH as a PLP-dependent enzyme that couples pyrroline-5-carboxylate, generated from L-ornithine, with L-aspartate to form (2S,5R)-5-((S)-2-amino-2-carboxyethyl)pyrrolidine-2-carboxylic acid (ACPCA), which corresponds to the nonpeptidic Ala-Pro substructure of KCP. D2O-labeling experiments showed enzyme-controlled, solvent-derived deuterium incorporation at C7 of ACPCA, supporting a decarboxylative Mannich-type mechanism. Feeding of a deuterium-enriched ACPCA-containing reaction mixture to the KCP-producing fungus Eupenicillium shearii resulted in deuterium incorporation into KCP, linking ACPCA to KCP biosynthesis. These results identify KpbH as the first native PLP-dependent enzyme that catalyzes an L-aspartate-dependent decarboxylative Mannich-type C-C bond-forming reaction and reveal a biosynthetic strategy for constructing a noncanonical amino acid-like C-C bond scaffold.

The systematic navigation of biocatalyst space is constrained by elusive structure-activity rules and a lack of evolutionary history. Here, we present IMUSE, a strategy integrating machine learning with ultra-high-throughput screening. By screening millions of droplet-encapsulated de novo enzymes, we generated massive synthetic sequence-structure datasets to train models that capture their complex fitness landscapes and biophysical principles. These models effectively guide functional exploration across both sequence and novel structure spaces. IMUSE identified synergistic triple mutations yielding ~5-fold activity improvements and discovered active second-generation designs with novel catalytic pockets, boosting the experimental success rate >4.9-fold (~30%). This work demonstrates how synthetic fitness landscapes bridge the data gap in de novo enzyme space, transforming stochastic search into deterministic navigation to unlock highly proficient biocatalysts beyond natural boundaries.

Nature Chemical Biology, Published online: 24 June 2026; doi:10.1038/s41589-026-02247-5

Cytochrome P450 enzymes are defined by a conserved cysteine ligand to the heme iron that is crucial for catalytic function. Now, the discovery of several distinct families of non-canonical P450s that lack this cysteine ligand, including naturally occurring serine- and selenocysteine-ligated enzymes, is reported, thereby challenging a longstanding paradigm in P450 enzymology.

Biocatalysis has become an important part of synthetic chemistry, particularly in the pharmaceutical industry. It is therefore important for chemistry students to learn about biocatalysis in their programs. We have developed an experiment for the second-...

Piperazate synthases (PZSs) catalyze the key N─N bond formation in the biosynthesis of the rare amino acid piperazic acid. X-ray crystal structures of apo- and holo-SbPZS show a dimeric enzyme with two heme-containing active sites, where histidine residues coordinate the iron centers. Structural analysis and molecular dynamics simulations provide insights into cofactor binding, active-site architecture, and enzymatic N─N bond formation.

l-Piperazic acid (l-Piz) is a noncanonical, α-hydrazino acid characterized by a 1,2-diazinane heterocycle containing an N─N bond. It occurs in numerous natural products with potent biological activities and represents a key pharmaceutical building block. In nature, l-Piz is biosynthesized from l-ornithine via the intermediate N ⁵-hydroxy-l-ornithine in a two-enzyme cascade comprising a flavin adenine dinucleotide (FAD)-dependent N-hydroxylating monooxygenase (NMO) and a heme-dependent piperazate synthase (PZS). The NMO selectively hydroxylates the δ-amino group of l-ornithine, while PZS catalyzes intramolecular N─N bond formation to generate the six-membered cyclic hydrazine scaffold of l-Piz. Here, we report the crystal structure, Piz-forming activity, and molecular dynamics (MD) analysis of SbPZS, a representative PZS from Streptomyces sp. B93. High-resolution structural analysis enabled a detailed comparison with previously characterized PZS homologs. To further delineate the molecular basis of catalysis, we performed MD simulations in combination with sequence-based bioinformatic analyses. These studies provide insight into protein–substrate interactions, conformational dynamics, and the residues that contribute to active-site organization. Moreover, we identify candidate hotspots for engineering to modulate substrate scope and catalytic efficiency. Collectively, our results establish a structural framework for understanding enzymatic N─N bond formation in Piz biosynthesis and lay the groundwork for future biocatalytic applications of PZSs.

Enzyme assays and computational analyses reveal that human histidine methyltransferases SETD3 and METTL9 have a capacity to catalyze histidine ethylation in the presence of synthetic or in situ produced S-adenosylethionine (AdoEth) and Se-adenosylselenoethionine (AdoSeEth) cosubstrates.

AdoMet-dependent histidine methyltransferases catalyze regioselective methylation of histidine residues in proteins. N^τ-Methylation of His73 in β-actin is catalyzed by histidine methyltransferase SETD3, and represents a unique post-translational modification involved in the regulation of actin polymerization. Likewise, N^π-methylation of His375 in zinc transporter SLC39A5 is catalyzed by histidine methyltransferase METTL9, thereby modulating zinc-binding properties of SLC39A5. Here, we report biomolecular studies on the ability of human SETD3 and METTL9 to catalyze the histidine ethylation reaction beyond methylation. Combined synthetic, biocatalytic and computational analyses employing synthetic or in situ formed AdoMet analogs AdoEth and AdoSeEth reveal that AdoMet is the most efficient cosubstrate; however, SETD3 and METTL9 also have the capacity to catalyze ethylation of histidine in β-actin and SLC39A5 peptides, respectively. Computational analyses support the experimental observations and provide the structural origin for more efficient histidine methylation than ethylation reaction. This work provides an insight into the molecular requirements for histidine methyltransferase-catalyzed histidine methylation and most related ethylation reactions on the N^τ- and N^π-positions in the imidazole ring, the knowledge important for functional assignment and design of chemical probes targeting histidine methyltransferases.

Nature Chemical Biology, Published online: 22 June 2026; doi:10.1038/s41589-026-02235-9

Cytochrome P450s catalyze essential reactions and carry a strictly conserved proximal cysteine ligand. Here, we identify noncanonical P450s that harbor diverse proximal ligands, including serine and selenocysteine, expanding the P450 chemical space and providing opportunities for future discoveries.

Nature, Published online: 18 June 2026; doi:10.1038/d41586-026-01947-1

Reliance on artificial-intelligence tools degrades the abilities of physicians and software engineers, studies show.

Nature Chemistry, Published online: 19 June 2026; doi:10.1038/s41557-026-02185-8

Few nickel enzymes have been discovered to date, limiting understanding of nickel-catalysed chemistry in nature. Now, a unique family of nickel pincer mononucleotide-utilizing enzymes has been identified through bioinformatics and characterized through biochemical and structural studies, expanding the known repertoire of nickel enzymes.

Acm11 is a distinct member of the glycoside hydrolase subfamily with dual catalytic functions. The enzyme not only hydrolyzes starch and oligosaccharide substrates, but also catalyzes the transglycosylation of carbasugar derivatives in the presence of maltooligosaccharides, yielding novel products with improved inhibitory activity against α-amylase.

ABSTRACT

Glycoside hydrolases (GHs) comprise a large and diverse family of enzymes that catalyze the hydrolysis of glycosidic bonds in a wide range of glycan substrates, including polysaccharides, oligosaccharides, and glycoconjugates. In addition to their fundamental roles in biological systems, GHs are widely exploited in industrial and biomedical applications. Here, we report the identification and functional characterization of a new GH, Acm11, from the biosynthetic gene cluster of pseudo-oligosaccharide acarviostatin I03 in a streptomyce strain. Bioinformatic analysis predicts Acm11 to be an α-amylase containing a C-terminal starch-binding domain. Disruption of the acm11 resulted in decrease of acarviostatin I03 production. Consistently, in vitro enzymatic assays demonstrated that Acm11 efficiently hydrolyzes starch, indicating its involvement in acarviostatin I03 biosynthesis. Notably, Acm11 also exhibited hydrolytic activity toward oligosaccharide substrates, progressively removing glucose units from acarviostatin I03 and acarbose. In addition to hydrolysis, Acm11 catalyzed C7-cyclitol-(1→4)-glycosylation of acarviostatin I03 and acarbose in the presence of maltooligosaccharides, revealing an unexpected transglycosylation activity. Mutational analysis indicated that distinct amino acid residues are responsible for hydrolytic and transglycosylation activities and evolution of Acm11 is expected to yield variants with altered catalytic preferences as the promising biocatalysts to generate structurally diverse and functionally improved α-amylase inhibitors.

Luciferases are widely used bioluminescent reporters, yet access to these enzymes has historically relied on recombinant expression. Here, we show that automated fast-flow peptide synthesis (AFPS) enables the rapid production of functional luciferases ...

Arginine side chains play important roles in the catalytic sites of many natural and engineered enzymes. This versatility is further exemplified by the recent development of artificial enzymes for SNAr processes, which rely on a key catalytic arginine ...

Metal–hydride hydrogen atom transfer (MHAT) reactivity is well established in homogeneous catalysis, but analogous reactivity in enzyme active sites remains rare and mechanistically underexplored. A recently discovered non-heme Fe/2-oxoglutarate (Fe/2OG) ...

Metal centers in enzymes are typically considered to operate within well-defined coordination geometries; however, emerging evidence suggests that dynamic metal behavior could also play a functional role. Herein, we report a structure-informed density ...

Science Advances, Volume 12, Issue 25, June 2026.

Science Advances, Volume 12, Issue 25, June 2026.

NADPH dehydrogenase gene deletion significantly triggered NADPH/NADP⁺ increase in mutant strains. NADPH gene deletion promoted Fatty acid biosynthesis and repressed β-oxidation. NADPH gene deletion decreased oxidative phosphorylation and increased substrate-level phosphorylation. Mutants exhibited hypersensitivity to oxidative stress, but promoted more fruiting body differentiations.

ABSTRACT

Nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase is an oxidoreductase involved in many physiological processes and metabolic pathways. However, its role in filamentous fungal physiology is still unclear. In the present study, three canonical NADPH dehydrogenase genes (Panph1, Panph2, and Panph3) in the fungus Podospora anserina were deleted, and multiple mutants were constructed. Results show a significantly increased number of fruiting bodies in the NADPH dehydrogenase mutant, with the nph ^ΔΔΔ triple mutant exhibiting higher sensitivity to oxidative stress, suggesting an active-site protein misfolding. Specifically, the antioxidant genes Nox and CAT in the WT were significantly down-regulated, confirming NADPH availability; however, in the NADPH mutant, these genes were significantly upregulated (p ≤ 0.001) as a response to nullify the constraint imposed by NADPH deletion and alleviate oxidative stress. Furthermore, an increase in substrate-level phosphorylation compensated for a significant decrease in oxidative phosphorylation due to NADPH gene deletion. The NADPH/NADP⁺ ratio, a driving force for the intracellular redox potential, showed a significant increase in the nph ^ΔΔΔ triple mutant. Meanwhile, the NADPH mutant inhibited the β-oxidative pathway, decreasing fatty acid degradation, but promoted fatty acid biosynthesis, reflecting the role of NADPH in the metabolic programming of cellular respiration and energy utilization processes in fungi. Our study provides genetic evidence for the role of the NADPH dehydrogenase gene in oxidative defence and energy metabolism in P. anserina.

Nature Chemistry, Published online: 17 June 2026; doi:10.1038/s41557-026-02184-9

There is limited understanding of nickel-catalysed chemistry in nature. Now a bioinformatic pipeline reveals a structurally distinct family of nickel pincer nucleotide-dependent enzymes. Nickel pincer hydride transferase (NphT) was found to catalyse intermolecular hydride transfer by characterizing its crystal structure, conducting structure-guided mutagenesis and measuring its capacity to disproportionate sugars.

Proceedings of the National Academy of Sciences, Volume 123, Issue 24, June 2026.
SignificanceComputational methods in biocatalysis are mostly focused on biocatalytic matrix engineering, whereas identifying the most reactive substrates for a given catalyst remains much less developed. By quantitatively reproducing the experimentally ...

We have developed a fluorometric assay for detecting reductase activity in biological samples through 4-methoxy-1-naphthalenemethanol (MONOL-41) formation. The enzyme carbonyl reductase 1 (CBR1) and four members of the aldo-keto reductase (AKR) 1 family (AKR1A1, AKR1B1, AKR1B10, AKR1C3) were evaluated for their ability to reduce 4-methoxy-1-naphthaldehyde (MONAL-41). AKR1B1 and CBR1 followed Michaelis-Menten kinetics, whereas AKR1B10, AKR1A1, and AKR1C3 showed substrate inhibition above 10 {micro}M (70 {micro}M for AKR1C3). Among the tested enzymes, AKR1B10 displayed the highest catalytic efficiency in the absence of substrate inhibition. The MONOL-41 assay was compared with the standard NADPH-based method, showing improved sensitivity, robustness, and lower detection limits (0.77 {micro}g/mL vs. 1.49 {micro}g/mL). These results confirm its suitability for monitoring AKR1B10 activity. The assay was then applied to A549 cell extracts, which express multiple reductases. Activity decreased at substrate concentrations above 10 {micro}M, suggesting a predominant role of AKR1B10. Inhibition studies using tolrestat and high MONAL-41 concentrations indicated a limited contribution of CBR1 ([~]7-8%). Considering both catalytic efficiency and expression levels, AKR1B10 appears to be the main contributor to reductase activity in this model. In A549 living cells, MONAL-41 showed no cytotoxicity up to 50 {micro}M and enabled real-time monitoring due to its membrane permeability. However, oxidation by aldehyde dehydrogenases can generate MONOIC-41, which has similar spectral properties but a lower quantum yield, potentially affecting signal interpretation. Overall, this assay represents a sensitive and cost-effective tool for detecting reductase activity and screening inhibitors.

Semi-rational engineering yields a thermostable Bacillus simplex formate dehydrogenase variant (Q125F) with a 25-fold longer half-life at 60°C and strong resistance to chemo-inactivation. Molecular dynamics reveal enhanced π–π interactions stabilizing a flexible loop and enabling efficient NADH regeneration for robust biocatalytic detoxification.

Formate dehydrogenase (FDH) catalyzes the reversible formate oxidation using NAD⁺ to yield carbon dioxide and NADH. FDH is widely utilized for cofactor regeneration, enabling the continuous supplying of NADH for chiral chemical biosynthesis. Herein, we employed semi-rational engineering to improve the thermostability of highly catalytic FDH from Bacillus simplex (BsFDH). Through two rounds of mutational screening, BsFDH^Q125F variant was identified to exhibit significantly improved thermostability with a 25-fold increase in half-life at 60°C. Additionally, the BsFDH^Q125F variant revealed significant stability against chemo-inactivation. The 100-ns molecular dynamics stimulation demonstrated a reduced overall root-mean-square deviation for the BsFDH^Q125F, with enhanced local packing primarily driven by intra- and inter-subunit π–π interaction among four tandem histidine and phenylalanine residues, thereby restricting the movement of flexible loop130−160. The implementation of the BsFDH^Q125F variant for cofactor regeneration in 4-nitrophenol detoxification demonstrated its superior stability and efficiency under bioconversion, rendering it suitable for various biocatalytic industrial applications.

Ribonucleotide reductases (RNRs) are valuable biocatalysts for the biosynthesis of non-natural deoxyribonucleotide, relevant in fields like medical science and synthetic biology. In this study, we explore the substrate spectrum of two thermostable RNRs and discuss their implications for nucleotide metabolism and biocatalytical applications.

Ribonucleotide reductases (RNRs) catalyze one of the central biochemical reactions, giving rise to deoxyribonucleotides, the building blocks of DNA. Due to their importance in cellular metabolism, this class of enzymes has been extensively studied over five decades. One aspect that has been neglected so far is the substrate specificity in terms of noncanonical nucleotides. While some of these compounds are physiologically relevant, many non-natural nucleotides are important in medical science, biotechnology and synthetic biology. In this study, we investigated the substrate specificity of two thermostable RNRs for a broad range of natural and non-natural nucleotides, in order to define the substrate promiscuity of this class of enzymes. Both enzymes were capable of converting all canonical nucleotides and a variety of other nucleotides. Generally, the enzymes were more likely to convert substrates with modifications of already existing functional groups of the nucleobase core structure. Our results show the potential and limitations for the biotechnological application of RNRs. In addition, they improve our understanding of the natural nucleotide metabolism in dealing with naturally occurring nucleotide analogues.

SAM-dependent enzymes are classified into S _N 2 methyltransferases, radical SAM enzymes, and nonmethylating enzymes. Their distinct mechanisms (S _N 2, radical, and nonmethylating reactions) and recent protein engineering strategies for improving activity, selectivity, and stability are summarized.

S-Adenosyl-_L-methionine (SAM) is a central biological cofactor that supplied activated methyl groups and enables a broad-spectrum biochemical transformation. Beyond canonical S _N 2 methyl transfer, SAM-dependent enzymes could initiate radical-mediated chemistry via reductive SAM cleavage, enabling versatile reactions from methyl transfer to alkylation, isomerization, and decarboxylation. Despite their extraordinary catalytic plasticity and potential for sustainable industrial synthesis, SAM-dependent enzymes are often limited by insufficient activity, stability, and substrate scope, necessitating further engineering. In this review, we summarize SAM-dependent enzymes into methylation and nonmethylation catalytic systems, and systematically summarize recent protein engineering efforts aimed at enhancing activity, selectivity, and stability. Together, these advances establish a unified framework for unlocking the full catalytic potential of SAM-dependent enzymes, paving the way for their integration as versatile and sustainable biocatalysts in modern chemical synthesis.

Protein kinases (PKs) are enzymes that catalyze phosphorylation of protein substrates involved in a wide variety of biological signalling pathways. This review describes the different families and mechanisms of action and regulation of protein kinases, together with the different classes of fluorescent biosensors that have been engineered and implemented to report on PK activities in vitro and for imaging purposes.

Protein kinases (PKs) are enzymes that catalyze phosphorylation of protein substrates involved in a wide variety of biological signalling pathways. PKs can be distinguished based on their mechanism of action and their regulation. They are associated with numerous pathologies, thereby constituting attractive biomarkers and targets for development of therapeutics. Given their importance, a wide variety of strategies and technologies have been devised for their detection, their quantification, and for studying their dynamic behavior, from radioactive and antibody-based approaches to biochemical, kinomic, and biosensing technologies.

A wide variety of fluorescent biosensors have been tailored to report on PK activities in vitro and for imaging purposes. These tools allow researchers to monitor PK activities in a highly sensitive and dynamic fashion in complex samples, providing new avenues for investigating PK behavior in native conditions and following stimulation or inhibition with drugs, thereby providing functional information which complements genetic, transcriptomic, or proteomic approaches. This review focuses on the different families of PKs and on fluorescent biosensors and chemosensors available to report, profile and image PKs from the AGC, CMGC, CAMK, TK, and TKL kinases.

Angewandte Chemie International Edition, EarlyView.

This review highlights recent advances in integrating biocatalysis and chemocatalysis in continuous flow to create streamlined, sustainable processes. It examines chemo-enzymatic cascades combining at least one enzymatic and one chemical step, discusses challenges such as enzyme immobilization, leaching, and reactor clogging, and presents solutions like enzyme engineering, reactor design, and automation that enhance compatibility and efficiency.

ABSTRACT

The growing demand for complex molecules continues to drive innovation in organic synthesis, yet challenges in sustainability, selectivity, scalability, and harsh reaction conditions persist. Enzymes offer exquisite chemo-, regio-, and stereoselectivity under mild conditions, while chemocatalysis provides robust and versatile reactivity. However, integrating these approaches into streamlined processes remains difficult due to incompatible conditions and operational constraints. Continuous flow chemistry offers a promising solution by enabling the efficient combination of biocatalysis and chemocatalysis, while improving atom economy, reaction control, scalability, and energy efficiency. This review highlights key advances up to 2025 in merging enzymatic and chemical steps into streamlined continuous flow cascades. It analyzes examples involving various enzyme classes—hydrolases, oxidoreductases, lyases, transferases, and isomerases—used alongside chemical catalysts. Major challenges such as enzyme immobilization, catalyst leaching, and reactor clogging are discussed, along with innovative solutions. The review also discusses how advanced enzyme engineering and immobilization strategies enhance biocatalyst activity, stability, and compatibility with chemical steps. By outlining recent progress and future directions, this review emphasizes how the integration of biocatalysis, chemocatalysis, and flow chemistry can foster more sustainable and efficient synthetic methodologies, particularly relevant to the pharmaceutical and fine chemical industries.

Biocatalytic metal-containing clusters are nanometer-sized chemical reactors that enable enzymes to perform chemistry far beyond what would be possible with amino acids alone 1,2. These clusters display a remarkable variability3 and elucidating their exact mechanisms is often difficult to determine, requiring input from a multitude of techniques ranging from spectroscopic to structural and theoretical methods. Thus, for unknown clusters, it is essential to accumulate, collate, and interpret as much information as possible from every relevant technique available. Here we report the discovery and in-depth, interdisciplinary characterization of an entirely novel, [Cu-4Fe-4S] cluster in the protein acetol dehydrogenase (AceDH). AceDH, isolated directly from Methylacidiphilum fumariolicum SolV cells, catalyzed the oxidation of acetol to methylglyoxal, proving its role in the 2-propanol/acetone metabolism of methanotrophs4-6. Structural, spectroscopic and electrochemical analyses reveal the [Cu-4Fe-4S] cluster has a unique three-dimensional- and electronic structure involving electronic coupling between the copper and one of the iron atoms, likely contributing to its high, +275 mV, redox potential. The binding site for the novel cluster is composed of two protein subunits and involves a novel motif. These findings expand the known repertoire of biological metal cofactors and provide insight into how heterometallic clusters are adapted for biological processes.

Organisms and their enzymes adapt to environmental temperatures, such that thermophilic enzymes exhibit high melting and optimum temperatures while psychrophilic enzymes exhibit low values for both. It has been proposed that the gap between an enzymes optimum temperature and its melting temperature, the temperature gap, is characteristically large in psychrophiles, implying that the loss of activity above the optimum is decoupled from global protein stability. The evidence for this relies on a small number of characterised enzymes, leaving the prevalence of large temperature gaps amongst psychrophiles unknown. We asked whether the machine-learning predictors and large datasets now available could test this at scale. We find that they cannot: predictors of melting and optimum temperature fail systematically at the thermal extremes, assigning the majority of thermophilic enzymes with optimum temperatures that exceed their melting temperatures, which is biophysically implausible, and consistently underpredict the stability of (hyper)thermophiles. This stems from training data that is both error-laden, as we demonstrate for widely used optimum-temperature records, and overwhelmingly biased toward mesophiles, which regresses predictions for cold and heat adapted enzymes toward mesophilic values. Consequently, current computational tools cannot establish how prevalent the psychrophilic temperature gap is. We argue that proteome-scale measurement of extremophile enzyme thermal behaviour, integrated as curated training data, is required to determine whether trends from small studies extend across the diversity of life.