Biocatalysis@TUDelft

The CoE-AmDH panel enables sustainable access to chiral amines using diverse amine (AmDH) and amino acid dehydrogenases (AADH). Utilizing formate (FDH) or glucose dehydrogenase (GDH) for cofactor regeneration, this study establishes a robust screening platform for mild reductive aminations. The panel rapidly identifies biocatalysts with tuneable stereoselectivity across broad substrate scope, enabling green biotransformations for enantiomeric small molecule synthesis.

ABSTRACT

In this study, we report the expression and characterization of a new panel of amine dehydrogenases (AmDHs) and amino acid dehydrogenases (AADHs). The panel of enzymes was initially screened against a library of candidate aldehyde and ketone substrates, in order to identify potential hits, using an NAD(P)H depletion assay. In some cases beneficial mutations from the literature were introduced to relevant sequences to improve activity. Finally some specific ketones (e.g., 17a, 18a, and 19a) were studied in detail in order to characterize the stereochemistry of the transformations.

NifB uses an RS–K1–K2 triad to assemble the L-cluster, a [Fe₈S₉C] precursor of the nitrogenase cofactor. The RS module cleaves SAM to form the 5′-dA radical, K1 serves structural/SAM-sensing roles, and K2 drives radical-dependent fusion with K1. SAM-anchoring residues link SAM binding to cluster fusion, while termini and surface residues regulate activity, collectively enabling efficient cluster formation.

NifB, a radical SAM enzyme, catalyzes the formation of a distinct [Fe₈S₉C] core (L-cluster) of the nitrogenase cofactor. Prior studies have led to the proposal of three [Fe₄S₄] modules—RS, K1, and K2—that mediate fusion of K1 and K2 via radical SAM chemistry at RS, but the identities and functions of cluster ligands and SAM-binding residues have remained unclear. Here, we report a systematic mutagenic analysis of key residues of NifB. Combining EPR spectroscopy with biochemical assays, we verify C18, H31, and C115 as ligands of the K1-cluster, and C260 and C263 as ligands of the K2-cluster. We further reveal a functional asymmetry in which the K1-module controls the initial sensing and orientation of SAM, whereas the K2-module acts as the catalytic center for radical-driven cluster fusion. Mutations in SAM-binding residues (T139, N194, P225) uncouple substrate binding from catalysis, while alteration of a surface residue (C240) enhances activity, implicating conformational gating in catalysis. Together, these findings define a functionally differentiated RS–K1–K2 triad and establish a mechanistic framework for radical SAM-dependent L-cluster assembly.

Schematic illustration of the workflow from DNA sequence-based research to expression in shaking flasks and subsequent purification via IMAC. The obtained enzymes broaden the toolbox of enzymes used for halogenation reactions with differing catalytic properties and tolerances to reaction-related conditions. This scheme is created with BioRender.com.

Halogenation reactions are essential for producing many fine and bulk chemicals. Vanadium-dependent chloroperoxidases (VCPOs) are promising biocatalysts for a more sustainable production of halogenated products because they are cofactor independent, use hydrogen peroxide (H₂O₂) as oxygen donor, tolerate high H₂O₂ concentrations, and catalyze all halogenations except fluorination. Despite their outstanding catalytic potential, few VCPOs have been identified yet, and detailed information on their catalytic characteristics is lacking. Here, we report the heterologous expression of three previously uncharacterized VCPOs in Escherichia coli and their validation. One of the enzymes was identified in the fungus Curvularia clavata (CcVCPO). The other two were found in the bacteria Crocinitomix catalasitica (CrocVCPO) and in an uncultivated Bacteroidetes strain (UbVCPO) isolated from a water sample taken from lake Neuchâtel in Switzerland. These enzymes exhibit highly attractive features, including thermostability up to 40°C for the bromination reaction of monochlorodimedone (MCD), the highest chlorination turnover numbers reported to date for wild-type VCPOs for the variant UbVCPO, as well as increased enzyme activation at 40°C and in the presence of methanol or ethanol for the same enzyme variant. These findings significantly expand the VCPO toolbox and highlight their potential for sustainable halogenation processes.

The immobilization performance of M2-32 varied with clay type, with palygorskite providing the highest catalytic reaction and stability. After immobilization, the enzyme preserved its broad pH and temperature tolerance. Neither the free nor the immobilized protein produced measurable changes in the rhizosphere microbial diversity. Instead soil type, plant presence and incubation time were the main determinants of microbial structure. These findings identify palygorskite-immobilized M2-32 as a robust, and environmentally compatible biocatlyst suitable for further application.

ABSTRACT

We explore enzyme-based technologies as sustainable alternatives to conventional chemical fertilisers, addressing the challenges associated with using enzymes in free or immobilised form for agricultural applications. We use the metagenome-derived Class A acid phosphatase M2-32, selected for its high activity, broad pH tolerance and thermophilic properties, and evaluated its immobilisation on clay minerals to enhance stability and applicability in soils. Several clays were tested as immobilisation supports. Bentonite caused complete enzyme inactivation, while kaolin formed aggregates and was unsuitable. In contrast, palygorskite, sepiolite and agrozeolite adsorbed more than 99% of the added enzyme. However, only a fraction of the immobilised enzyme retained catalytic activity, with optimal performance observed at moderate protein loading (40–80 μg protein). Among the tested supports, palygorskite consistently provided the highest specific activity (22,000 ± 2200 U/mg), followed by sepiolite (11,000 ± 730 U/mg), whereas agrozeolite (2250 ± 40 U/mg) showed comparatively low activity. ATR-FTIR spectroscopy confirmed successful enzyme immobilisation without significant alteration of the clay structures. Immobilised M2-32 preserved a broad pH range (between 4 and 8.5) and thermophilic behaviour similar to the free enzyme, remaining active up to 50°C. Immobilisation increased substrate affinity while reducing V_max relative to the free enzyme. To assess environmental compatibility, the effects of free and palygorskite-immobilised M2-32 on soil microbial communities were evaluated using corn rhizosphere microcosms with different organic matter contents. Metabarcoding high-throughput sequencing revealed that microbial diversity and community structure were primarily shaped by soil type, plant presence and incubation time. Enzyme application, whether free or immobilised, did not significantly alter microbial diversity or composition. Overall, these results support palygorskite-immobilised M2-32 as a promising, environmentally compatible candidate for enzyme-based fertiliser development.

The de novo design of enzymes critically tests our understanding of natural enzymes and enables design of novel catalysts. Here, we identify the features responsible for the catalytic efficiency of a highly proficient de novo enzyme generated through computational design and optimized by directed evolution. Computational, spectroscopic, and biochemical studies reveal successfully designed features, including precise alignment of catalytic residues, transition state stabilization, and environmental tuning. In the most evolved enzyme, the binding of a transition state analog also led to widespread increases in backbone rigidity and conformational stability throughout the protein, except within a helix near the active site entrance, where the introduction of Gly and Pro increased dynamics and catalytic activity. Thus, the entire protein contributes to catalysis in the most optimized enzyme. These studies provide principles for designing efficient enzymes.

Old Yellow Enzymes (OYEs) are a widely distributed family of ene-reductases that were first described in a Saccharomyces cerevisiae ferment. In plants, cis-12-oxo-phytodienoic acid (cis-OPDA) reductase (OPR) is the best studied OYE. In Arabidopsis thaliana, the peroxisomal AtOPR3 was characterized as the major OPDA reductase, which generates 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid in the jasmonic acid (JA) biosynthesis. In Atopr3 lines, only small amounts of JA are detectable after wounding. Here, we describe an OPR-like enzyme (named BnOPR) from the gram-positive Brevibacillus nitrificans. The sequence was identified in an early version of the Physcomitrium patens genome and is assumed to be a contamination by a bacterium growing in association with P. patens. In complementation experiments with an Atopr3 line, we demonstrate that expression of BnOPR, fused with a peroxisomal targeting signal, rescues the male infertile phenotype and increases JA and JA-Ile levels. The catalytic parameters of BnOPR were determined for a set of substrates, including cis-OPDA and prednisone. Interestingly, B. nitrificans, B. brevis, and Paenibacillus physcomitrellae were shown to have a positive effect on P. patens growth.

Active-site redesign frequently yields modest improvements because residues controlling physical steps like substrate binding and product release lie outside the active site. Efficient catalysis requires a cooperative catalytic network of residues that support both the chemical and physical steps of catalysis. Using ancestral hydroxynitrile lyase HNL1, an /{beta}-hydrolase with poor esterase activity, we tested this framework directly. Matching all active-site residues to a proficient esterase improved KM five-fold but left kcat unchanged, confirming that chemical machinery alone is insufficient. Activity-weighted sequence comparison (SigniSite) across ten homologous HNLs and esterases identified 38 positions disfavoring esterase activity. Experimental refinement yielded a minimal set of fifteen substitutions (HNL1-15) with ~60-fold higher kcat and ~400-fold higher kcat/KM. Single-substitution reversion analysis confirmed that all fifteen substitutions are essential and provided evidence for strong cooperativity between them. X-ray crystal structures of HNL1 and HNL1-15 reveal three coordinated structural changes: reshaping the substrate-binding pocket to favor productive ester binding, restoring access to the oxyanion hole, and opening an additional tunnel for product egress and water entry. These changes arise through backbone rearrangements and altered flexibility rather than direct active-site contacts, explaining why the responsible positions escape conservation-based detection. Because cooperativity masks individual contributions, engineering such networks may require step-specific assays - measuring binding, acylation, or product release directly - rather than screening composite kcat.

Our current industrial, agricultural, and medical practices exploit the extraordinary biodiversity generated through billions of years of natural evolution. Despite their high fitness in native habitats, biomolecules and organisms are often not optimally suited for industrial and medical use. Synthetic evolution leverages technologies such as DNA synthesis, CRISPR–Cas engineering, and synthetic biology to enable continuous in vivo mutagenesis of biomolecules or organisms to improve specific desirable characteristics. This review presents the latest mutagenesis toolkits classified by mutational scale: genome-wide, medium-scale, and site-specific, each tailored to different application scenarios. We discuss the mechanisms and capabilities underlying each scale, analyze current limitations, and highlight the untapped potential of next-generation gene-editing technologies, high-throughput screening, and artificial intelligence in advancing synthetic evolution.

Nature Communications, Published online: 30 May 2026; doi:10.1038/s41467-026-73764-z

Chloromethane, a toxic gas primarily produced naturally, contributes to ozone destruction. Here, the authors identify and characterise an enzyme system that dehalogenates chloromethane and appears to be specific to anaerobic microorganisms.

Nature Biotechnology, Published online: 29 May 2026; doi:10.1038/s41587-026-03158-5

Radio waves are shown to modulate fluorescence and associate spin chemistry in proteins.

Nature Chemical Biology, Published online: 29 May 2026; doi:10.1038/s41589-026-02231-z

We developed ROProx, a blue-light-activated, H2O2-free proximity labeling technology that uses the chemical probe BN2 and leverages the intrinsic tyrosyl radical of the peroxidase APEX2. Taking advantage of a dual-brake mechanism, ROProx enables rapid capture of dynamic protein complexes in cultured cells within seconds and within minutes in vivo.

An Artificial metalloenzyme was designed by a Michael addition between a MnSalen complex and a cysteine residue within NikA crystals. The heterogeneous catalyst was found capable of an enantioselective and stereospecific epoxidation of cis-β-methylstyrene in cristallo.

ABSTRACT

Artificial enzymes represent a promising alternative for performing non-natural reactions in biocatalysis. Here, we illustrate the potential of cross-linked enzyme crystals (CLEC) to achieve enantioselective epoxidation through the generation of an artificial enzyme obtained by direct covalent anchoring of a manganese complex as an artificial active site within a protein. Enantiomeric excess (ee) of up to 90% on cis-β−methylstyrene was measured when the covalent binding yield was maximized, thanks to the remarkable behavior of the crystals. The structure of the modified enzyme, NikA, is provided. This work adds to the growing body of examples highlighting the advantages of CLEC in oxidation catalysis.

Despite the difference in active site architecture, NO reduction by different NOR enzymes is expected to be linked by a common intermediate: hyponitrite (N₂O₂ ²⁻). However, experimentally, very little is known about the coordination chemistry of iron with hyponitrite and the expected Fe-hyponitrite intermediates. In this Perspective, we provide a detailed discussion of Fe-hyponitrite chemistry, and comparison with select Ni- and Cu-hyponitrite complexes.

ABSTRACT

Nitric oxide reductases (NORs) are a class of metalloenzymes that are known to mediate the 2 e⁻/ 2 H⁺ reduction of nitric oxide (NO) to nitrous oxide (N₂O). There are three known types of NOR enzymes, each using a different iron-based active site to mediate NO reduction. Despite the difference in active site architecture across NORs, NO reduction is expected to be linked by a common intermediate: hyponitrite (N₂O₂ ²⁻). However, experimentally, very little is known about the expected Fe-hyponitrite intermediates. In this Perspective, we provide a detailed discussion of Fe-hyponitrite chemistry. We start with an introduction into NOR metalloenzymes and provide a detailed account of the known and proposed steps of catalytic NO reduction by these enzymes with a specific focus on the proposed Fe-hyponitrite intermediates. We then turn our focus on what is known about synthetic Fe-hyponitrite complexes. We introduce hyponitrite as a ligand and discuss sources of preformed hyponitrite. We then introduce the three known Fe-hyponitrite complexes and discuss how these relate to NO reduction in NORs. Finally, we summarize what is known about Fe-hyponitrite chemistry and give an outlook on the aspects of this chemistry that are still vastly underexplored.

Bridged or not? ⁵⁷Fe NRVS reveals a non-covalent, electronically coupled interaction between the HydF [4Fe–4S] cluster and the [2Fe] precursor. Boltz-2 structure predictions suggest that the cubane organizes lipoate-dependent H_met chemistry, positioning intermediates for assembly of the CH₂–NH–CH₂ bridge during H-cluster biosynthesis.

ABSTRACT

Maturation of [FeFe]-hydrogenase depends on the HydF maturase, a [4Fe–4S]-containing scaffold protein that assembles and delivers the organometallic [2Fe] subsite of the H-cluster. Despite extensive research, the function of the HydF [4Fe–4S] cluster and its interaction with the [2Fe] cofactor remains unresolved, with conflicting evidence regarding cyanide linkage isomerism and the functional role of the cubane. Using ⁵⁷Fe nuclear resonance vibrational spectroscopy in combination with selective isotopic labeling, we show that the [2Fe] subsite binds adjacent to the [4Fe–4S] cluster without forming a covalent cyanide bridge, while remaining electronically coupled. Complementary protein structure predictions support this arrangement and reconcile prior spectroscopic, mutagenesis, and structural studies. Extending this framework to earlier biosynthetic steps, additional structure predictions suggest that the [4Fe–4S] cluster contributes to assembly of the CH₂–NH–CH₂ bridge of the [2Fe] site via interactions with the lipoate cofactor of the aminomethyl-lipoyl H-protein, thereby positioning reactive components in proximity to the [2Fe] precursor. Together, these results provide a coherent structural and mechanistic framework for HydF function across distinct stages of H-cluster biosynthesis.

Nature Chemical Biology, Published online: 29 May 2026; doi:10.1038/s41589-026-02233-x

To obtain the semi-essential amino acid cysteine, cells assimilate its oxidized form, cystine, and then reduce its disulfide bond. By genetically deleting the disulfide reductase pathways from mouse liver, we uncovered an inducible, ubiquitous, cell-autonomous reductase-independent backup pathway for the generation of cysteine, driven by cystine C–S bond cleavage.

Allenes and alkynes are versatile functional groups in total synthesis, medicinal chemistry, and bioorthogonal conjugation. The biosynthetic logic of how Nature installs allene or alkyne in natural products, especially that of allenes, is not well understood. Here we uncovered allenes and alkynes can be formed enzymatically through oxidative C(sp2)-demethylation of the common five-carbon prenyl group. Two fungal cytochrome P450 monooxygenases, PpnB and NseB, from the penipratynolene and sinuxylamide biosynthetic pathways, respectively, were shown to catalyze oxidative removal of a C(sp2)-methyl group in O-prenyl-L-tyrosine to afford O-homoallenyl-L-tyrosine and O-but-2-ynyl-L-tyrosine, respectively. Combining density functional theory calculations, heterologous expression, biotransformation and enzymatic assays with isotopically labeled substrates, a mechanism involving selective C-C bond cleavage followed by product-determining hydrogen atom abstraction is presented. An additional P450 enzyme from the penipratynolene pathway, PpnD, acts as an oxidative isomerase that converts the four-carbon terminal allene into a terminal alkyne. This unprecedented enzymatic editing strategy to install allene and alkyne expands the catalytic repertoire of P450 enzymes.

Nucleotide-detected solid-state NMR has been applied to probe nucleotide conformations and dynamics during ATP hydrolysis using the dimeric P-loop ATPase SmsC as a model protein. The different stages of ATP hydrolysis have been mimicked by ATP analogues for which different degrees of conformational heterogeneity have been observed.

ABSTRACT

Solid-state nuclear magnetic resonance (NMR) spectroscopy is an ideal tool to study ATP hydrolysis in ATP-driven processes at atomic resolution. In this study, we present a nucleotide-detected solid-state NMR approach to probe nucleotide conformations and dynamics during ATP hydrolysis using the dimeric P-loop ATPase SmsC as a model protein. ³¹P-detected solid-state NMR experiments under magic-angle spinning (MAS) conditions provide insights into the different stages of ATP hydrolysis, which have been mimicked by ATP analogues that are often considered as “nonhydrolyzable”. Our data reveal that different degrees of conformational heterogeneity are observed for such ATP mimics. By computational modeling, we explore the role of a hydrogen bond between SmsC and the terminal phosphate group of the nucleotides in defining the nucleotide binding conformation. Homonuclear ³¹P-³¹P dipolar coupling constant measurements for the bound ATP analogues have been performed to obtain information about their dynamic properties. These data show that the ATP and the transition-state analogues maintain some molecular motion, while the post-hydrolytic mimic AMPCP shows significantly less dynamics. The approach presented herein for investigating ATP hydrolysis can be easily transferred to other ATP-fueled proteins, including difficult to express proteins or large protein complexes, therefore providing a way to functionally characterize these systems.

The nitrosuccinate lyase CreD catalyzes C–NO₂ bond formation using nitrite in water and shows synthetic practicality with high turnover numbers up to 102,000. A combination of protein engineering and computational methods helped to reveal the mechanistic principles that underpin this unique enzymatic activity.

ABSTRACT

Nitro compounds are central to synthetic chemistry, yet mild and selective biocatalytic routes to these motifs remain elusive. We report an enzymatic strategy for the unique decarboxylative hydronitration of fumarate, achieved by repurposing the nitrosuccinate lyase CreD from the aspartase/fumarase superfamily for the synthetic direction. CreD from Streptomyces cremeus and three related bacterial homologues catalyze hydronitration using sodium nitrite salt with remarkable efficiency (turnover numbers up to 102,000) and high atom economy. With an already exceptionally broad functional group tolerance across the superfamily, this feature underscores a conserved yet adaptable activity landscape. Guided by comprehensive mutagenesis and computational analysis across all functionally distinct superfamily members, we uncovered key molecular determinants that govern nucleophile selectivity and preserve the structural integrity required for active tetramer assembly. We also define a diagnostic fingerprint for predicting hydronitration activity and propose a reaction mechanism supported by extensive QM/MM simulations. These molecular insights provide a foundation for expanding biocatalytic Michael-type additions under environmentally benign aqueous conditions.

Yeasts are promising cell factories, yet metabolic rigidity limits their biosynthetic potential. By developing a host-specific framework integrating pathway rewiring, regulator reprogramming, and fermentation optimization, P. pastoris was engineered to produce the fragrance sclareol at 27.8 g l−1, providing a scalable blueprint for bio-based manufacturing.