Biocatalysis@TUDelft

Lignin is a promising alternative to petroleum as a feedstock for the chemical industry. Emergent strategies for lignin valorization involve tandem processes in which biomass is chemo-catalytically fractionated followed by biotransformation of the depolymerized lignin by microbial cell factories. A rate-limiting step in this biotransformation is O-demethylation of the lignin-derived monomers. The reductive catalytic fractionation of hardwood biomass generates high yields of two classes of monomers: 4-alkylguaiacols and 4-alkylsyringols. To better understand the biotransformation of these monomers, we studied AgcA, a cytochrome P450, and AgcB, the cognate reductase, that together catalyze the O-demethylation of 4-alkylguaiacols. A 1.82 [A] resolution crystal structure of AgcAEP4 from Rhodococcus rhodochrous EP4 in complex with 4-ethylguaiacol identified residues Leu78, Ala293 and Phe166 as potential specificity determinants. Substitution of Ala293 and Leu78 decreased the specificity of AgcAEP4 for alkylguaiacols. Substitution of Phe166 yielded a variant that bound 4-propylsyringol but did not transform it. In contrast, the corresponding variant in the Rhodococcus aromaticivorans RHA1 homolog, AgcARHA1 Y166A, catalyzed the O-demethylated of both methoxy groups of 4-propylsyringol with a kcat/Km of 8500 M-1 s-1 for the first O-demethylation, nearly 7-fold higher than WT AgcARHA1. A strain of RHA1 harboring the variant did not grow on 4-propylsyringol but consumed it at approximately the same rate as 4-propylguaiacol and transformed some of it to pentanoyl-CoA, consistent with metabolism via the meta-cleavage pathway that catabolizes 4-alkylguaiacols. These studies improve our understanding of a critical lignin-degrading enzyme system and facilitate its efficient implementation into biocatalysts. SignificanceLignin is a highly abundant source of aromatic carbon and a promising alternative to petroleum to generate materials. Fulfilling this promise depends on technological advances in areas such as catalytic fractionation and biocatalysis. Catalytic fractionation of hardwood biomass generates mixtures of aromatics enriched in 4-propylguaiacol and 4-propylsyringol. Here, we biochemically and structurally characterized a cytochrome P450 that initiates 4-propylguaiacol catabolism. Informed by the structure, we engineered the enzyme to have dual activity on both 4-propylguaiacol and 4-propylsyringol, and implemented this enzyme into a bacterial biocatalyst. Metabolomic analysis of this strain provided insights into the catabolism of both aromatics. Overall, these findings greatly facilitate the engineering of P450s and bacteria to biocatalytically upgrade lignin.

Hyaluronic acid (HA) is a biologically versatile polysaccharide synthesized by vertebrates and several microbial pathogens. To date, Cryptococcus neoformans CPS1p is the only reported hyaluronic acid synthase (HAS) in fungi, which is functionally related to bacterial HASs. Considering the phylogenetic and biochemical connection between chitin synthases (CHSs), essential for fungal cell wall synthesis, and HASs, it is reasonable to hypothesize the latter might be more common in fungi than expected. In this work, a comprehensive in silico survey of putative HASs in the Fungal Tree of Life was carried out. 68 putative HASs, mainly in Basidiomycota, were found, although other AI-inferred HASs were found among Ascomycota. Global fold and arrangement of essential amino acids were shared by all kingdoms HASs; however, fungal HASs showed additional exclusive conserved sequence signatures. Moreover, fungal HASs bore an only 3-helices transmembranal pore and their gating loop, which regulates the entrance of substrates to the catalytic site, was directly connected to an also exclusive intrinsically disordered (IDR) C-terminus. Phylogenetically, fungal HASs were found in a clade different to that of bacterial, animal and viral HASs, and all HASs shared the same ancestor with class VI CHSs. The atypical features of fungal HASs could influence the size and biological role of the HA they synthesize and also highlight potential regulatory differences among HASs at the gating loop configuration level. ImportanceDespite the report of CPS1p, the hyaluronic acid synthase (HAS) of Cryptococcus neoformans, the diversity, structural features and biochemical assets of fungal HASs remain unknown. Here, 68 putative fungal HASs were identified, mainly among Basidiomycota. Although their fold is similar to that of already characterized HASs, their transmembranal pore, integrated by only 3 helices, and their atypical gating loop configuration, suggest they could be also differently regulated, influencing size and function of HA they synthesize.

Nature Chemistry, Published online: 23 February 2026; doi:10.1038/s41557-025-02052-y

Most H2 used in the chemical industry is derived from fossil fuels. Now it has been shown that coupling native microbial H2 pathways with engineered alkene biosynthesis and membrane-bound Pd catalysis enables biocompatible hydrogenation of metabolic intermediates in living bacteria. This hybrid chemo-microbial platform supports the carbon-negative synthesis of industrial chemicals from waste-derived feedstocks.

Engineered hemoproteins enabled the stereoselective C–H amination of carboxylic esters to access enantioenriched aryl-β-amino esters. Directed evolution yielded variants with excellent chemoselectivity towards the use of O-pivaloylhydroxylamine triflic acid or hydroxylamine hydrochloride as aminating reagents. Through the collection of partial sequence-activity data, beneficial mutations to improve the final variants catalytic activity were identified.

ABSTRACT

Engineered biocatalysts can utilize nitrene precursors to access enantioenriched amination products, yet they have not been applied to produce valuable, enantiomerically enriched noncanonical β-amino esters. Current approaches to synthesizing β-amino acids rely on pre-oxidized precursors and multistep synthetic approaches involving various protecting groups. We engineered a platform of heme enzymes for stereoselective C–H bond amination of readily available carboxylic ester derivatives to install primary amines. A directed evolution campaign coupled with sequencing of over 1000 variants enabled us to develop engineered variants that use either O-pivaloylhydroxylamine triflic acid (PONT) or hydroxylamine hydrochloride (H₂NOH∙HCl) as aminating reagents. An analysis of the resulting sequence–activity dataset revealed additional improvements that could be made to the final variant, highlighting the utility of sequencing data to guide future steps in directed evolution campaigns. The evolved nitrene transferases expand the scope of accessible chiral β-amino acid building blocks for peptidomimetic applications and provide new starting points for the design and synthesis of enantioenriched β-amino acid motifs.

Nature Communications, Published online: 21 February 2026; doi:10.1038/s41467-026-69586-8

Predicting chemical reactions remains a challenge. Here, the authors present Loxodynamics, a machine learning method that uses statistical skewness to automatically discover reaction pathways in complex systems without prior knowledge.

Yu and co-workers have advanced the development of living materials for biocatalysis by reporting a platform based on a 3D-printable hydrogel matrix. Within this matrix, phase-separated aqueous microdroplets compartmentalize distinct microbial consortia, thus facilitating a stable redox balance and enabling continuous-flow biocatalysis.

ABSTRACT

Constructing living materials with microbial consortia represents an emerging approach for creating life-like functional systems; however, the spatiotemporal orchestration of the activities across diverse species remains a major challenge. Yu et al. address this with a 3D-printable hydrogel matrix that embeds phase-separated aqueous microdroplets for microbial compartmentalization, enabling sustained biocatalysis under continuous flow (https://doi.org/10.1002/anov.70015).

A minimal protein, asparaginase (AdePA), is characterized. We report the first experimental crystal structure for this enzyme class, revealing a novel fold and a Ser-His-Asp catalytic triad indicative of a serine protease-like mechanism. This compact enzyme efficiently catalyzes thess deamidation of protein Asn to Asp residues and exhibits high thermal stability, overcoming the limitations of previously known large, unstable PAs.

Enzymatic deamidation of proteins, catalyzed by protein glutaminase (PG) for Gln or by protein asparaginase (PA) for Asn residues, is a key strategy for improving functional properties such as solubility and foaming. However, the only known PA, from Luteimicrobium album (LalPA), is a large, thermally unstable multidomain protein (1355 aa) that has proven challenging to express heterologously. To overcome these limitations, we identified a novel, compact PA from Amycolatopsis deserti (AdePA) using a comprehensive database search. We then solved the first experimental structure of any PA, which revealed a catalytic mechanism utilizing a Ser-His-Asp catalytic triad indicative of a serine protease-like function, which is distinct from that of L-asparaginase. AdePA offers significant advantages over LalPA; it is a smaller (785 aa) single-domain enzyme with superior thermal stability (retaining 50% activity at 40°C, where LalPA is inactivated) and is readily produced through heterologous expression. Furthermore, AdePA shows inverted substrate specificity, preferring sterically small N-terminal groups, making it highly effective for modifying unstructured proteins like gelatin. These findings demonstrate that AdePA is a robust candidate for industrial applications in protein modification.

Applied and Environmental Microbiology, Volume 92, Issue 3, March 2026.

Nature Communications, Published online: 19 February 2026; doi:10.1038/s41467-026-69812-3

Microbial cells have emerged as a versatile platform for the synthesis of metal nanoparticles, but their application to produce single-atom catalysts (SACs) has been rarely studied. Here, the authors develop a facile method for the ambient synthesis of SACs with a high loading by in situ reduction of metal ions on the cells overexpressing a catalytically active enzyme, producing chemo-bio bifunctional catalysts (SAC@cell).

The amine transaminase-catalyzed amination of (R)-3-hydroxyphenylacetylcarbinol toward metaraminol involves severe side-product formation, hampering high product titers. The application of a continuous extraction system together with an increased amine transaminase concentration and optimal enzyme formulation enhanced the overall metaraminol yield.

Metaraminol is a chiral amino alcohol and plays an important role as a precursor molecule and active pharmaceutical ingredient in industry. Its enzymatic synthesis has been developed in recent years and can serve as an alternative to conventional synthesis routes that use toxic, fossil-based resources. Although the enzymatic two-step reaction toward metaraminol has been intensively investigated in the past, full conversion has never been reached in the amine transaminase-catalyzed step. In this study, we focus on identifying and overcoming the hurdles of the transamination step to reach higher metaraminol yields. Photometric and LC-MS analyses revealed side-product formation as a major drawback for the enzymatic metaraminol synthesis. Besides the oxidation of (R)-3-OH-PAC as well as its imine formation with isopropylamine, we demonstrate for the first time the adduct formation of the cofactor pyridoxal-5’-phosphate with metaraminol. Only by changing the amine transaminase formulation to purified enzyme and increasing the concentration by tenfold, >99% product yield with a metaraminol concentration of 75 mM was reached. Further, we successfully integrated the amine donor l-alanine by applying a continuous product extraction system as an alternative to isopropylamine. We believe that our findings and optimization strategies can also serve as a blueprint for other amine-based syntheses.

For decades, structure–function has dominated biochemistry. Structures are highly valuable, yet more is needed to achieve a quantitative understanding of biomolecular function, because function emerges from an ensemble of states, rather than a static structure. We describe an ensemble–function framework applied to quantitatively dissect serine protease catalysis. This framework provides quantitative mechanisms of biological functions grounded in basic chemical and physical principles, an approach that can transform biochemical research and education.

In this perspective, we describe how we arrived at a framework of ensemble–function analyses to quantitatively dissect enzyme catalysis and biological function more broadly. Serine proteases are described in biochemistry textbooks to illustrate enzyme mechanisms, yet those descriptions do not explain how these enzymes achieve their ~ 10¹²-fold rate enhancements. Moving away from the classic descriptions of ‘catalytic triad’ and ‘oxyanion hole’, we returned to the basic physical and chemical interactions in serine protease active sites and identified molecular features that enable a highly efficient reaction path on the enzymes, compared to the uncatalyzed reaction. We then leveraged principles from statistical mechanics to quantify the contributions from each catalytic feature. Combining the contributions from each feature in a ‘catalytic ledger’ provided a quantitative accounting of serine protease catalysis. These analyses revealed previously unrecognized catalytic interactions that are destabilizing in the reaction's ground state—unfavorable bond rotamers, shorter-than-ideal distances, and suboptimal hydrogen bonds—each of which is relieved in the transition state, thereby lowering the barrier to reaction. Analogous catalytic features are found in over 30 different protease and nonprotease enzymes spread across 12 structural folds, suggesting that nature has taken advantage of these strategies multiple times in different contexts. In the future, ensemble–function analyses can be used to derive quantitative mechanistic models for other enzymes, to dissect allostery, and to ascertain how molecular machines operate. Ensemble–function also provides a powerful educational approach by linking the complex behavior of biomolecules to the simple chemical and physical principles that are taught in undergraduate classes.

Nature Chemical Biology, Published online: 18 February 2026; doi:10.1038/s41589-026-02172-7

Striking the balance in enzyme evolution

Communications Chemistry, Published online: 17 February 2026; doi:10.1038/s42004-025-01876-6

Cytochrome P450 monooxygenases can functionalize alkaloid scaffolds with striking stereo- and regioselectivity. Here, the authors integrate bioinformatics and enzyme discovery to identify P450 monooxygenases capable of selectively functionalizing anticancer alkaloid evodiamine, revealing their catalytic activities and potential application in diversifying pharmacologically important compounds.

Nature Catalysis, Published online: 12 February 2026; doi:10.1038/s41929-026-01478-y

Predicting the function of enzymes remains difficult and current computational methods require improvement. Now EnzymeCAGE, a geometric deep learning model, has been developed to more accurately predict the functions of uncharacterized enzymes and reconstruct biosynthetic pathways.

Ribonucleases are widely regarded as degradative enzymes, but the reversibility of RNA cleavage also enables ligation through cyclic phosphate intermediates. Here, RNase T₁ is repurposed as a catalyst for RNA dynamic covalent chemistry, driving the thermodynamically controlled recombination of short strands into stable hairpins. The reaction is highly selective and functions under diverse conditions, revealing a constructive role for RNases in programmable RNA assembly.

Dynamic covalent chemistry (DCC) provides a powerful framework for assembling complex molecular architectures under thermodynamic control. Here, we extend RNA DCC by harnessing the ability of RNase T₁—traditionally used as a degradative enzyme—to catalyze reversible phosphodiester exchange. Although enzyme-mediated RNA ligation has been reported previously, such reactions typically required high substrate concentrations and yielded heterogeneous mixtures without structural control. By coupling catalysis to RNA folding, selective formation of well-defined hairpin products governed by thermodynamic stability was achieved. PAGE, LC-MS, and NMR analyses confirm high-fidelity ligation at low temperatures with yields up to 61%, directed by loop stability and stem complementarity. Four distinct RNA oligomers assemble into two hairpins in one pot without cross-ligation, demonstrating RNA DCC as a programmable strategy for equilibrium RNA assembly. This work outlines an RNase-catalyzed framework for structure-guided RNA recombination, showcasing an underexplored pathway for the ligation of folded RNA polymers.

Apicomplexan parasites like Toxoplasma gondii harbor a highly divergent mitochondrial proteome, much of which remains uncharacterized despite its essentiality for parasite survival. One such critical pathway is ubiquinone (UQ) biosynthesis. Here, we characterize the UQ synthesis machinery in T. gondii and show that conserved enzymes, TgCoq3 and TgCoq5, are essential for growth and mitochondrial function, forming a multi-protein complex. Using proximity labeling and subcellular fractionation, a strategy suited for detecting proteins of low abundance, we identify TgCoqFAD, a unique FAD-dependent monooxygenase required for UQ synthesis. Unlike canonical eukaryotic systems that employ multiple monooxygenases to modify specific carbons on the UQ aromatic ring, TgCoqFAD catalyzes two distinct hydroxylation steps, an activity not previously reported in eukaryotes. Molecular docking and chemical screening identified TgCoqFAD inhibitors that impair tachyzoite growth and bradyzoite viability. These findings reveal a streamlined and divergent UQ biosynthesis pathway in apicomplexans and establish TgCoqFAD as a promising antiparasitic target.

Nature Synthesis, Published online: 12 February 2026; doi:10.1038/s44160-026-00998-6

Biophotoelectrocatalysis-driven oxyfunctionalization of C–H bonds is often hampered by unselective photoelectrochemical water oxidation and the generation of peroxygenase-deactivating hydroxyl radicals. Here a bicarbonate redox mediator redirects water oxidation from a direct pathway to an indirect pathway for H2O2 formation that avoids radical stress, enabling robust oxygenative biosynthesis.