Biocatalysis@TUDelft

IsdG is a member of the Staphylococcus aureus iron-regulated surface determinant system that degrades haem scavanged from a human host to a mixture of staphylobilin and biliverdin; the rate-limiting step of this enzyme is the rearrangment of a ferric–peroxohaem species to a hydroxylated ferryl=oxohaem speices. In this work, the role of conserved Asn and Trp residues in catalysing this reaction are interrogated by preparing and characterizing ferryl haem analogues for the hydroxylated ferryl=oxohaem speices in N7A and W67F IsdG. The ferryl haem form of N7A IsdG had an extremely short lifetime, and could only be detected by lowering the temperature to 5 °C. In contrast, the ferryl haem form of W67F IsdG could be observed at room temperature, but the lifetime of the ferryl haem form of W67F IsdG (110 s) was less than half that reported for wild-type (WT) enzyme (250 s). Magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) characterization of the reaction mixture between ferric heme-bound N7A IsdG and meta-chloroperoxybenzoic acid (mCPBA) did not yield any evidence for a ferryl haem species, consistent with the extremely short lifetime of the ferryl haem form of N7A IsdG. Similarly, clear evidence for a ferryl haem form of IsdG was not obtained upon MCD characterization of the reaction mixture between the ferric haem form of W67F IsdG and mCPBA. However, EPR characterization of the W67F IsdG reaction provided clear evidence for the presence of a compound I-like ferryl haem species distinct from the compound ES-like ferryl haem reported for WT enzyme. The lifetime measurements revealed that both Asn7 and Trp67 have important roles in stabilizing the ferryl haem form of IsdG, with Asn7 making a more significant contribution. Furthermore, the correlation between organic radical location in the ferryl haem form of IsdG and the ratio of staphylobilin to biliverdin generated by the enzyme suggests that this organic radical may play a major role in determining the product selectivity of IsdG.

Gibberellins are structurally complex diterpenoid plant hormones with widespread agricultural applications. However, of the 136 known congeners found in nature, very few are easily accessible, preventing further research regarding their bio-logical function. In biosynthesis, oxidases from plants generate the diverse oxidation patterns of natural gibberellins. Plant oxidases, in contrast to microbial oxidases, are very challenging to use for chemoenzymatic synthesis. Here, we develop a chemoenzymatic and metabolic engineering platform using plant oxidases that allows the production and full characterization of eight rare gibberellins as well as 16 ent-kaurene derivatives with various oxidation patterns. The fila-mentous fungus Aspergillus oryzae was engineered to produce common gibberellins and pathway intermediates in high titers, resulting in a panel of 20 diterpenoid substrates. These were screened using leaf disks from the model plant Nico-tiana benthamiana producing ten different plant oxidases from diterpenoid metabolism. From the 200 substrate-enzyme pairs a total of 65 compounds were identified. We scaled up five reactions to produce milligram quantities of oxidized diterpenoids. Finally, by adding one of the plant oxidase genes to A. oryzae, we also accessed 15β-hydroxylated gibberel-lins in a single step by metabolic engineering. In summary, our work enables the flexible and sustainable synthesis of rare gibberellins and other highly oxidized diterpenoids. More importantly, our work demonstrates how plant oxidases can be used in chemoenzymatic synthesis and total biosynthesis campaigns, which will help to better utilize the catalytic potential of these previously neglected enzymes in the future.

Nature Synthesis, Published online: 27 November 2025; doi:10.1038/s44160-025-00963-9

Author Correction: Discovery and engineering of the biosynthesis of rotenoids

A dynamic kinetic resolution approach is developed for the atroposelective synthesis of heterobiaryl primary amines. Using transaminases and leveraging Lewis acid-base interactions to induce racemization, a variety of axially chiral primary amines are produced in high yields and enantioselectivities. This mild, metal-free method expands the scope of biocatalytic asymmetric synthesis.

Atropisomeric heterobiaryl primary amines are of significant interest in both organic and pharmaceutical chemistry. A series of transaminases have been employed to synthesize these valuable compounds with high yields (up to 98% conversion) and excellent enantioselectivities (up to ≥99% ee) via dynamic kinetic resolution of the corresponding heterobiaryl aldehydes. This process features a Lewis acid–base interaction strategy to facilitate labilization of the stereogenic axis.

Proceedings of the National Academy of Sciences, Volume 122, Issue 47, November 2025.
SignificanceTriglycerides (TAGs), the primary form of long-term energy storage, have acyl chain compositions crucial for diverse cellular processes. Lipases typically hydrolyze TAGs into free fatty acids. Here, we reveal a function for the neutral lipid ...

Proceedings of the National Academy of Sciences, Volume 122, Issue 47, November 2025.
SignificanceCooperativity in protein function is critical for the ability of all organisms to respond effectively to environmental changes. Various mechanisms have been proposed to underlie such cooperativity, but they are almost always invoked and ...

Substrate channeling is a strategy for enhancing flux and yield in enzymatic cascades and is increasingly relevant for applications in biocatalysis, biotechnology, and bioelectrochemical systems. However, efforts to engineer channeling are limited by the lack of high-throughput methods to evaluate and optimize channeling efficiency. Here, we present a fluorescence-based screening assay to rapidly assess substrate channeling in a model system involving fumarase and malate dehydrogenase, two sequential enzymes from the Krebs cycle. By expressing genetic fusions in E. coli, quantifying intermediate (malate) and product (NADH) formation in lysate using orthogonal fluorescent readouts, and comparing product-to-intermediate ratios, we screened a library of linker variants designed to promote electrostatic channeling. A top-performing construct was identified and validated through classical channeling assays. This hit demonstrated increased product yield and current output when immobilized on electrodes with a bilayer architecture, highlighting utility in bioelectrocatalysis. We further showed that the channeling linker could be applied to a de novo designed single-chain fumarase, which preserved channeling capability and exhibited improved thermal stability. These results establish a generalizable and scalable method for engineering and evolving substrate channeling, with broad implications for pathway optimization and enzyme design in synthetic biology, bioprocessing, and energy applications.

Kinetic and MD methods were used to examine the catalytic mechanism of the triazine hydrolase (TrzN) from Arthrobacter aurescens TC1. Plots of the pH dependence of k _cat, K _m, and k _cat/K _m revealed a pK' _ES < 4.5, a pK _ES > 11, and a pK _E value of 6.4 ± 0.2. Proton inventory studies indicated that at least three protons are transferred in the rate-limiting step. MD combined with temperature studies reveal enthalpically driven reactions.

The catalytic mechanism of the Zn(II)-dependent triazine hydrolase (TrzN) from Arthrobacter aurescens TC1 was examined by measuring the pH dependence of the Michaelis constants k _cat, K _m, and k _cat/K _m, the solvent isotope effect, and the thermodynamic parameters of the hydrolysis of atrazine. TrzN was maximally active towards atrazine over the pH range 6.5–10.0, and fits of these data yielded a pK' _ES < 4.5, a pK _ES > 11, and a pK _E value of 6.4 ± 0.2. Based on these data, along with those previously reported, the observed pK' _ES and pK _E values are likely due to the active site residues Glu241 and His274, whereas the observed pK _ES value is possibly due to Tyr215. Proton inventory studies indicated that at least three protons are transferred in the rate-limiting step of the reaction at pD 7.5. An Arrhenius plot was constructed from 278 to 308 K by plotting ln(k _cat) vs. 1/T, providing an E _a of 16.7 ± 0.3 kJ·mol⁻¹ and ΔH‡ and ΔS‡ values of 14 ± 2 kJ·mol⁻¹ and −170 ± 10 J·mol⁻¹ at 25 °C, respectively, resulting in a ΔG‡ of 65.5 ± 0.1 kJ·mol⁻¹. These data coupled with molecular dynamics simulations of wild-type TrzN and the TrzN Glu241Gln mutant provided evidence for the proposed catalytic roles of active site residues, and identified molecular motions associated with substrate binding and allosteric regulators of transition-state arrangement. Taken together, these data support the proposed catalytic mechanism for the hydrolytic dehalogenation of atrazine by TrzN.

Nature Synthesis, Published online: 20 November 2025; doi:10.1038/s44160-025-00940-2

A dual-cofactor artificial metalloenzyme is developed, incorporating a biotinylated nickel complex and a Strep-tagged peptide catalyst in adjacent streptavidin-binding sites. This synergistic artificial metalloenzyme achieves enantiodivergent Michael addition reactions with tunable stereochemistry and high turnover numbers across diverse ketone and enal substrates.

Nature Chemical Biology, Published online: 21 November 2025; doi:10.1038/s41589-025-02077-x

Radical FeII/α-ketoglutarate-dependent halogenases are powerful biocatalysts for C–H functionalization. Here, the authors reveal the mechanistic basis for chemoselectivity in a lysine halogenase.

In this study, we present a sustainable, one-pot, chemoenzymatic procedure for the synthesis of alkenes from the corresponding alcohols or amines in aqueous medium. The procedure is based on the laccase-TEMPO-mediated oxidation of substrates and subsequent Wittig reaction. The method has a number of advantages in terms of sustainability, such as application of water as the reaction medium, use of molecular oxygen as final oxidant, or formation of water as a byproduct of the oxidation step.

A general, chemoenzymatic one-pot procedure for the transformation of alcohols or amines to the corresponding alkene products in aqueous media has been reported. The procedure is based on the laccase-TEMPO-mediated oxidations of substrates and subsequent Wittig reaction. In this way, a one-pot sequence of two consecutive reactions has been developed, which has a number of advantages such as (a) no need of purification of the intermediate products, (b) application of water as green solvent and an enzyme-laccase as natural catalyst, (c) application of molecular oxygen as final green oxidant, and (d) formation of water as a byproduct of the oxidation step.

We show how engineered ancestral amide bond synthetases (ABSs) together with cofactor recycling using a single polyphosphate kinase paves the way for chemoenzymatic synthesis of pharmacophores. The figure illustrates how ancestral ABS has a more flexible C-terminal domain, which is associated with enhanced catalytic performance and expanded amine scope. An ancestral enzyme can thus be robust but still show enhanced interdomain mobility.

Amide bond formation is a basal transformation in synthetic chemistry and the pharmaceutical industry that is traditionally performed under harsh conditions, using excess amounts of amine and relying on coupling agents. Biocatalysis shows great potential in contributing to milder and more sustainable amide bond formation in water, in particular using the emerging family of amide bond synthetase (ABS) enzymes. Here, we use molecular dynamics, biocatalysis, and enzyme engineering to study amide bond formation in extant and ancestral ABS from Marinactinospora thermotolerans (McbA). Our results show that while being more thermostable, the C-terminal domain that delivers the amine substrate to the adenylated acid intermediate is more flexible in ancestral McbA, presumably leading to an extended amine scope as observed experimentally from a small panel of aliphatic and aromatic substrates. An engineered ancestor of McbA harboring a single mutation that presumptively represent a catalytic shift residue when going from ancestral to modern biocatalyst, show two to ten-fold improved conversions over its ancestral template while maintaining high thermostability, highlighting ancestral sequence reconstruction as a potent method in protein engineering. Kinetic experiments showed that the engineered ancestral enzyme had 2-fold higher apparent k _cat values in amide formation compared to extant enzyme, concomitant with relaxed substrate inhibition and loss-of-dependency on magnesium. Finally, we optimize ATP recycling utilizing a single polyphosphate kinase to showcase how engineered ancestral McbA together with reaction optimization is amenable for pharmacophore synthesis at a preparative scale.

Atropisomeric scaffolds are important building blocks in natural products, organocatalysts, metal ligands, and functional materials. This review introduces current developments for synthesizing atropisomers employing biocatalytic kinetic resolution, dynamic kinetic resolution, and desymmetrization strategies.

ABSTRACT

The asymmetric synthesis of atropisomers has garnered extensive attention in recent years. Atropisomers constitute a key structural motif in natural products, chiral ligands, organocatalysts, and functional materials. Despite progress driven by transition-metal and organocatalysis, inherent limitations in enantioselectivity and sustainability have hampered further development in this field. Alternatively, biocatalysis offers a promising solution employing strategies including (dynamic) kinetic resolution, desymmetrization, and other strategies. These biocatalytic processes operate under mild, environmentally friendly conditions, achieving high stereoselectivity that is often difficult to attain with traditional methods. This review highlights recent advances in the biocatalytic synthesis of atropisomers and offer insights in the development of the relevant field.

A compact, genetically encoded photoenzyme, miniSOG, enables spatiotemporally controlled bioorthogonal deboronative hydroxylation of diverse organoboronates in live cells by producing localized superoxide radical anions (O₂•⁻), establishing a versatile platform for manipulating fundamental cellular pathways. More in the Research Article (e15137) by Yiyun Chen and co-workers.

Conformational interchange of enzyme–substrate complexes are intrinsic to catalysis, yet their transient nature makes it elusive for understanding the substrate conformational dynamics. In the Research Article (e15778) by Yi-Lei Zhao, Hui-Lei Yu, and co-workers a “flipped” substrate binding conformation in the hydroxynitrile lyase from Prunus communis (PcHNL5) is revealed. Crystallographic and computational analyses show that this non-productive state must reorganize before catalysis proceeds. Mutagenesis destabilizes this non-productive conformation, promoting conversion to the productively competent state and markedly enhancing enzymatic efficiency.

Local electric fields guide protons toward the heme through two distinct electrostatic funnels at the γ- and δ-edges in heme peroxidases. Water-mediated pathways enable proton exchange with key residues such as Arg38 and His42. These findings reveal that nature pre-organizes electrostatic funnels and solvent channels to provide multiple, well-defined routes for proton delivery, conserved across heme peroxidase enzymes. More in the Research Article (e202515743) by Reynier Suardíaz, Emma L. Raven, Adrian J. Mulholland, and co-workers.

We reveal the high temperature crystal structure of a hyperthermophilic (Pyrococcus furiosus) rubredoxin at 393 K (120 °C), together with multiple complementary structures down to 100 K. The results are compared with molecular dynamics calculations. Significant changes in H-bonding are observed. Discussions about high-temperature protein structure and stability need to recognize that low temperature structures may not represent the high temperature case.

Abstract

How does the structure of a protein change as the temperature is raised from cryogenic conditions at 100 K to 393 K? Understanding the structure and dynamics of proteins under environmental extremes is relevant for human health, biotechnological applications, and our search for life elsewhere in the universe. Here we reveal the high temperature crystal structure of a hyperthermophilic (Pyrococcus furiosus) rubredoxin at 393 K (120 °C), together with multiple complementary structures down to 100 K. The results are compared with molecular dynamics calculations. Significant changes in H-bonding are observed. Discussions about high-temperature protein structure and stability need to recognize that low temperature structures may not represent the high temperature case.

Angewandte Chemie International Edition, Volume 65, Issue 2, 9 January 2026.

The dehydroxylation of catechols represents an important chemical transformation facilitated by gut bacteria in mammals. This reaction is catalyzed by pyranopterin molybdenum enzymes that belong to the DMSO reductase family. Despite their chemical and biological significance, the structure and mechanisms of catechol dehydroxylases remain uncharacterized. In this manuscript, we interrogated the active site structure of hydrocaffeic acid dehydroxylase from the gut bacterium Gordonibacter urolithinfaciens (Gu Hcdh) using Mo K-edge X-ray absorption near-edge structure (XANES) spectroscopy and extended X-ray absorption fine structure (EXAFS) analyses. In the oxidized state, the Mo(VI) ion is coordinated by a terminal oxo atom, a cysteine thiolate, and four sulfur atoms from the two bidentate pyranopterin dithiolene (PDT) ligands. Upon reduction to the Mo(IV) state, the active site remains hexacoordinate with a similar first coordination sphere, but the terminal oxo ligand present in the Mo(VI) state has been protonated to yield a coordinated hydroxyl ligand. The EXAFS-derived coordination geometries for the Mo(VI) and Mo(IV) sites are consistent with the results of bond valence sum (BVS) analyses. Reaction coordinate computations suggest the likely role of an active site carboxylate in facilitating substrate dearomatization and product formation. Protein sequence analysis and site-directed mutagenesis experiments reveal that Cys157 is ligated to the Mo ion, and Asp210 serves as a catalytically essential active site acid-base. Together, these analyses enrich our understanding of an emerging pyranopterin molybdenum enzyme family from the human gut microbiota.

The N-methyltransferase SgPsmC was characterised and applied in the kinetic resolution (KR) of easily obtainable pyrroloindolines. Substitution at 3a-position proved to be a principal determinant for the enzyme's enantioselectivity. While moving toward laboratory scale, the reaction was coupled with a methionine adenosyl transferase for in situ generation of S-adenosyl methionine, allowing the KR to proceed without any background reaction.

Abstract

Many natural products and pharmaceutical compounds bear the pyrroloindoline scaffold, highlighting the importance of the heterocyclic motif. Here, we aim at expanding the toolset for the selective synthesis of pyrroloindolines by characterising and employing the N-methyltransferase SgPsmC from Streptomyces griseofuscus, an enzyme involved in the biosynthesis of physostigmine, in a selective kinetic resolution of pyrroloindolines performed at a laboratory preparative scale.