Biocatalysis@TUDelft

The Cover illustrates the use of cGAS, a well-studied enzyme, as a promising biocatalyst for industrial applications. A bioprocess plant with a characteristic bioreactor represents scalable production, in which the enzyme cGAS catalyzes the formation of the cyclic dinucleotide cGAMP. The spotlight on the product molecule underscores the shift from fundamental biochemical research towards process development and biotechnological application. The image reflects the core theme of the Review by Makram Fataeri and Katrin Rosenthal (DOI: 10.1002/cbic.202500942) bridging the gap between molecular understanding and process-oriented implementation.

Under enzymatic action, the ADP-ribosyl group of NAD⁺ can be transferred to the phosphate termini of DNA/RNA and the amino acid residues of proteins. Through chemical synthesis, NAD⁺ can undergo ADP-ribosylation reactions with alcohols, azides, and p-nitrophenol.

Nicotinamide adenine dinucleotide (NAD⁺), as an endogenous donor for ADP-ribosylation, can modify DNA, RNA, and proteins, thereby participating in the regulation of the functions of these biomacromolecules. NAD⁺ serves as a reactant in both enzymatic and chemical synthesis. By employing a well-designed reaction process, the synthetic route can be significantly streamlined, enabling the preparation of structurally complex bioactive molecules in a step-saving and highly effective manner. This article reviews the latest research progress in this field. In the field of enzymatic synthesis, a strategy based on the HPF1/PARP1 complex has been developed. Earlier study shows that the recombinant HPF1/PARP1 complex can ADP-ribosylate a variety of substrates in vitro. In the field of chemical synthesis, the focus is on ionic liquid-mediated ADP-ribosylation reactions with controllable α/β configurations of products. These reactions help prepare biologically active ADP-ribosylated (ADPr) peptides from NAD⁺ and commercially available peptides. In addition, this article also outlines the applications of functional NAD⁺ derivatives in enzyme activity analysis and inhibitor development and discusses the challenges faced in this field, such as bio-compatible reaction conditions, synthesis for precise structural control, and structure–activity relationships between stereochemistry and biological functions of more ADPr derivatives.

Engineering of water capture in terpene synthases remains very challenging. We identified two regions in the selinadiene synthase active site pocket that can influence deprotonation or water capture in the final catalytic step and demonstrate a designed-in switch in product outcome. These insights can be used for engineering other sesquiterpene synthases to generate new biocatalysts.

Sesquiterpene synthases catalyse cyclisations and rearrangements of farnesyl diphosphate to produce a diverse array of sesquiterpenes generated by depronotation and/or water capture. However, the precise mechanisms and dynamics controlling the fate of the final carbocationic intermediate are not well understood. In our previous study, we engineered water capture in selina-4(15),7(11)-diene synthase (SpSdS) to produce selin-7(11)-en-4-ol as a major product at pH 6.0 by point mutation (G305E) in the K_helix region. To develop a more generalised protocol for this functional switch in sesquiterpene synthases, we identified and characterised a novel selina-3,7(11)-diene synthase (AsSdS) from Actinacidiphila soli through multiple sequence alignments which naturally contains glutamate at position 305 (E305). Through site-directed mutagenesis, creating variant G221T, we were able to instigate water capture in AsSdS to produce selin-7(11)-en-4-ol. Our findings identified two crucial regions in the active site pocket of selinadiene synthases: G/E305 in K_helix and T/G221 in H_helix, that have a reproducible effect on product outcome determination. We demonstrate that subtle, yet predictable changes to these residues impact the water capture as well as deprotonation capability of selinadiene synthases and this solvation aspect can be further exploited to engineer other terpene synthases to generate biocatalysts with unique product profiles for diverse applications.

Applied and Environmental Microbiology, Ahead of Print.

A metal-dependent class II KDSG aldolase that catalyzes the key C─C bond cleavage in the sulfosugar breakdown (sulfoglycolytic Entner–Doudoroff) pathway is structurally and biochemically defined. Crystal structures and kinetic analyses reveal a hexameric, Co^{2 +}/Mn^{2 +}-activated enzyme with sulfonate selectivity, establishing the molecular basis of sulfoglycolytic pyruvate formation.

ABSTRACT

Sulfoquinovose (SQ) is a major biogenic sulfonated sugar whose degradation fuels microbial sulfur and carbon cycling. In the sulfoglycolytic Entner–Doudoroff (sulfo-ED) pathway, 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) is cleaved by KDSG aldolase to yield pyruvate and sulfolactaldehyde, yet the structure and mechanism of this enzyme have remained unclear. We report the biochemical and structural characterization of a metal-dependent KDSG aldolase from Pseudomonas putida using chemo-enzymatically synthesized KDSG. The enzyme forms a homohexamer, with a (β/α)₈ TIM-barrel monomer assembling as a ‘dimer-of-trimers’. The enzyme exhibits optimal activity in the presence of Co²⁺ or Mn²⁺, consistent with other class II aldolases. Kinetic analysis revealed millimolar-range K _M values for KDSG and modest cross-reactivity with the related glycolytic intermediate, 2-keto-3,6-deoxy-6-phosphogluconate (KDPG). Crystal structures of the apo and Co²⁺•pyruvate-bound forms (2.85 Å and 2.80 Å) show a metal-coordinated active site at the subunit interface, with conserved residues mediating metal binding and catalysis, providing insights into the mechanism of sulfonate-specific aldol cleavage. Sequence-similarity network and genome-context analyses show that KDSG aldolases are widespread among Proteobacteria and typically cluster with sulfo-ED pathway genes. These results define the structural and mechanistic basis of KDSG aldolases and inform on their roles in bacterial sulfur metabolism.

Nature, Published online: 13 March 2026; doi:10.1038/d41586-026-00767-7

Tightly packed molecular chains can start to wiggle in many directions when an enzyme introduces energy.

Nature Chemistry, Published online: 13 March 2026; doi:10.1038/s41557-026-02084-y

The site-specific incorporation of noncanonical amino acids (ncAAs) has so far been limited to single-type ncAA incorporation in mammalian cells. Now, the repurposing of rare codons and engineering of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs enable up to five distinct ncAAs in a single protein, which can be applied to study mammalian pathways of interest.

Unidirectionally communicating, out-of-equilibrium DNA circuits are used to control enzyme activity. Transient DNAzyme activation generates fuel for temporal trypsin activation. Cyclic fuel-driven hybridization and exonuclease-mediated reset enable precise control over lifetimes, which is analyzed using combined experimental and computational approaches.

ABSTRACT

Unlike most synthetic systems, life constantly reorganizes itself through the irreversible consumption of energy-rich molecules and exhibits dynamic functionalities governed by spatiotemporally controlled biocatalytic processes. Inspired by this, we herein demonstrate unidirectionally communicating, out-of-equilibrium DNA circuits that enable network-guided control of the biocatalytic activity of an enzyme. The unidirectional communication is realized through the programmed, dissipative manipulation of information transfer. In this process, transient activation of a DNAzyme generates the fuel required for the temporal activation of trypsin. Prior to establishing this information-transfer framework, we employed fuel-driven dissipation to autonomously and temporally regulate the activity of nucleic acid and protein-based enzymes, each operating in individual cycles. The transient state of the systems is attained through rapid hybridization of DNA strands, while digestion of the DNA fuel by exonucleases regenerates the initial equilibrium state. These processes proceed in a cyclic manner, allowing the systems to attain an out-of-equilibrium state. Precise control over the lifetime of this transient state was achieved by regulating external factors, such as DNA fuel and exonuclease concentrations, and internally by exploiting the toe-hold length-dependent digestion kinetics of the exonucleases. To establish our findings, we adopted a combined approach that includes both experimental and computational methodologies.

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of Nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

The substrate scope of tryptophan synthase from Salmonella enterica (SeTrpS) was probed against 28 synthetic indole derivatives bearing aryl rings, N-methylamino groups, alkynes, and halogens, accessing a broad range of nonnatural l-tryptophans on analytical scale. Representative preparative scale reactions afforded 7-ethynyltryptophan and 5-phenyltryptophan in 25%–58% yield, demonstrating scalability.

ABSTRACT

Tryptophan derivatives are valuable non-canonical amino acids widely used as precursors for the synthesis of bioactive molecules, as biophysical probes or introduced into polypeptides to improve or create entirely new functions. The enzyme tryptophan synthase (TrpS) provides a sustainable biocatalytic route to access these compounds directly from substituted indoles and l-serine. In this work, the substrate scope of Salmonella enterica tryptophan synthase (SeTrpS) was probed using a diverse library of synthetic indole derivatives bearing aryl, N-methylamino, alkynyl, and halogen substituents. Notably, SeTrpS displayed moderate to high conversions for most of the indoles tested, particularly for those substituted at positions 6 and 7. Two representative preparative scale reactions demonstrated the scalability of the process, affording the corresponding l-7-ethynyltryptophan and l-5-phenyltryptophan analogues in 58% and 25% yield, respectively, and excellent purity. This study contributes to illustrate the versatility of SeTrpS as a biocatalyst for the efficient and sustainable synthesis of structurally diverse tryptophan analogues, expanding the known substrate tolerance of this enzyme.

Studies with naturally occuring variants of the Pseudomonas savastanoi ethylene-forming enzyme (PsEFE) inform on the effect of residues on turnover and product ratio. Crystallographic studies imply a potential for metal ion sensing.

Pseudomonas savastanoi pv. phaseolicola PK2 employs an Fe(II)-dependent ethylene/succinate-forming enzyme (PK2 PsEFE) to produce ethylene from 2-oxoglutarate (2OG). Here we report NMR-based assays showing that the putative P. savastanoi pv. glycinea PsEFE, which differs from PK2 PsEFE by a single residue, and the P. savastanoi pv.1449B PsEFE, which differs from PK2 PsEFE by 28 residues and a C-terminal 13-residue truncation, catalyze ethylene production from 2OG. Like the PK2 PsEFE, they catalyze oxidation of naturally occurring 2OG derivatives to give alcohol and diacid products. Crystallographic analysis demonstrates that the overall fold and active site of 1449B PsEFE is similar to that of PK2 PsEFE. Interestingly, 2OG was observed to adopt an atypical inverse metal ion binding mode in complex with 1449B PsEFE:Mn in which its 2-oxoacid group is positioned to interact with the guanidinium group of R277, but not the Mn ion, which substitutes for catalytically active Fe(II). Together with reported crystallographic results, this observation indicates that 2OG metal ion binding modes and conformations at the active sites of 2OG oxygenases can vary, possibly in a functionally or disease relevant manner.

Production of d-amino acids with the HcLAAO4– d-TA _Ei –CAT enzyme cascade. _l ‑amino acids are oxidatively deaminated by HcLAAO4 consuming molecular oxygen to the corresponding α‑keto acids. Subsequently, d-TA _Ei catalyzes the reductive amination of the keto acids using d‑alanine as co-substrate to produce the d‑amino acids and pyruvate. Catalase (CAT) was used to remove hydrogen peroxide (H₂O₂) generated by the LAAO to prevent potential side reactions.

ABSTRACT

The synthesis of enantiomerically pure d-amino acids is of growing interest due to their applications in pharmaceuticals, agrochemicals, and as building blocks in fine chemicals. While chemical synthesis methods are often environmentally unfriendly, enzymatic cascades offer a greener and more selective alternative. In this study, a one-pot enzymatic cascade was established consisting of the soluble l-amino acid oxidase from Hebeloma cylindrosporum (HcLAAO4), a catalase, and a (R)-selective transaminase from Enterococcus italicus (d-TA _Ei ) for the conversion of l-amino acids into the corresponding d-enantiomers. The cascade efficiently produced a range of d-amino acids including phenylalanine, methionine, histidine, and tryptophan in enantiomerically pure form. Optimization of enzyme concentrations revealed that balanced loading of HcLAAO4 and d-TA _Ei achieves high conversions of up to 93% with >99% enantiomeric excess. Co-immobilization of the enzymes on glutaraldehyde-functionalized hexylamine (HA_GA) beads enhanced catalytic efficiency and speed, resulting in up to 80% conversion. Moreover, immobilization significantly affected the pH range of both d-TA_EI and HcLAAO4. These findings demonstrate the potential of combining broad-substrate LAAOs with d-specific TA for sustainable and preparative-scale production of d-amino acids.