Biocatalysis@TUDelft

We revealed the crystal structure of eKatE from atypical E. coli. eKatE exhibits a hydrogen-peroxide-sensitive major channel, a stabilized dimeric interface, an unusual covalent bond between C392 and Y415, and other distinctive features. These structural features contribute to its enhanced catalase activity compared to KatE in typical E. coli. Consequently, eKatE demonstrates high ROS resistance, effectively lowering the surrounding ROS levels.

Catalase is a crucial enzyme that protects organisms from reactive oxygen species (ROS)-induced oxidative stress. eKatE, a recently identified catalase variant in commensal Escherichia coli (E. coli), significantly contributes to infectious diseases and inflammatory bowel disease (IBD). Here, we enhanced the ROS detoxification capacity of eKatE, distinguishing it from the typical E. coli catalase KatE. eKatE forms a tetramer with a well-folded N-terminal arm and a dual conformation of the long R173^eKatE, in contrast to the disordered N terminus and A173^KatE of KatE. Additionally, a V256-induced bottleneck in the major channel enhances the sensitivity of eKatE to H₂O₂, differing from A256^KatE. Furthermore, K294^eKatE flipped inside to shield the major and lateral channels more effectively than K294^KatE. Covalent bonding of C392^eKatE to the essential Y415 increased the catalytic activity compared with that of H392^KatE. Finally, the electrostatic potential surface of the eKatE tetramers differed from those of KatE, particularly near the substrate-inlet and product-outlet regions. These findings on the improved catalytic capacity of eKatE highlight its potential application in mitigating ROS-related diseases and treating IBD.

Science, Volume 389, Issue 6761, Page 690-690, August 2025.

Science, Volume 389, Issue 6761, Page 741-746, August 2025.

Science, Volume 389, Issue 6761, Page 711-715, August 2025.

Science, Volume 389, Issue 6761, Page 732-735, August 2025.

Thioxanthone is an efficient visible-light photosensitizer and facilitates reactions that are not known from natural enzymes. It is now possible to incorporate thioxanthone as a noncanonical amino acid (thioX) into proteins by an engineered amino acyl tRNA synthetase. With this approach we created a photoenzyme that catalyzes the photo-E/Z isomerization of a hydroxycinnamate ester.

Abstract

Photocatalysis in biocatalytic systems provides a promising approach for achieving selective and efficient chemical transformations under mild conditions. Naturally occurring photoactive cofactors are rare. To overcome this limitation, genetic code engineering can be applied to equip proteins with additional functionalities beyond those known in the 20 canonical amino acids. Here, we report the engineering of an aminoacyl-tRNA synthetase (thioXRS) that allows the incorporation of a thioxanthone-bearing noncanonical amino acid (thioX). As proof-of-concept, we utilized the versatile biocatalyst LmrR as a protein scaffold. We identified an active variant able to catalyze the E/Z-photoisomerization of a cinnamate ester derivative into coumarin. The reaction design allows direct monitoring through fluorescence measurements, as the fluorescent substrate is converted into a non-fluorescent product. This work demonstrates that thioXRS is a versatile tool for the future development of new-to-nature photoenzymes, expanding the synthetic capabilities of biocatalysis towards light-driven transformations.

The fluidized bed reactor combines the properties of a stirred tank reactor and a continuous tubular reactor. Its advantages are its high transfer capacity and its use with liquid and gas phases. This device has been used to degrade contaminants and synthesize cosmetics, food, pharmaceuticals, lubricants, and biodiesel compounds. Its versatility will constitute an attractive alternative for developing biocatalytic systems.

The main objective of this article is to review previous contributions on the applications of fluidized bed reactors (FBR) in biocatalysis. FBR combines the properties of a stirred tank reactor and a continuous tubular reactor, making it an efficient system for carrying out enzymatic reactions with immobilized enzymes. This equipment's advantages include its high transfer capacity and versatility, as it can be used with liquid and gaseous phases. According to the literature, these devices have been primarily used to degrade contaminants, synthesize cosmetic ingredients, produce food and pharmaceutical compounds, and synthesize biolubricants and biodiesel. The enzymes most used in fluidized bed mode are laccases, lipases, and proteases immobilized on methacrylate resins, mesoporous silicas, alginate, and chitosan beads. Enzyme immobilization is essential, as it can promote the suspension of biocatalyst particles, thereby increasing yields and productivity. One of the leading prospects for these systems is to stabilize the fluidized bed using a magnetic field and the concept of “microfluidization,” which enables the stabilization of smaller biocatalyst particles with smaller equipment, thereby increasing efficiency and intensifying the biocatalytic process. In the future, the versatility of FBR will constitute an attractive alternative for developing biocatalytic systems.

A novel synthetic route to generate oligonucleotides containing a monoadduct or γ-interstrand crosslink from a DNA-distorting mitomycin is presented. The oligonucleotides are thoroughly characterized using mass and circular dichroism spectroscopy as well as thermal denaturation studies. The structure of this novel crosslinked duplex is compared with that of less-distorting mitomycin ICLs using Molecular Dynamics simulations.

Mitomycin C (MC) is a powerful chemotherapy agent currently used in clinics for the treatment of various types of cancer. MC functions by inhibiting cellular growth through the formation of cytotoxic interstrand crosslinks (ICLs). These ICLs induced by MC have minimal impact on the DNA backbone, preserving its B-DNA structure. Recent research suggests that the cellular machinery recognizes and repairs ICLs differently based on their specific structure. To better understand how DNA distortion caused by MC ICLs influences cytotoxic effects, Herein, a novel mitomycin ICL is synthiesized that, unlike MC, significantly distorts DNA and widens the minor groove. This work outlines the synthesis of oligonucleotides bearing a single monoadduct or a single ICL of this new MC derivative at a defined position. Such substrates are widely used for investigations into biological processes such as DNA damage/repair studies. The monoadducted and crosslinked oligonucleotides are thoroughly characterized using various techniques, including enzymatic digestion to nucleosides, mass and circular dichroism spectroscopy, as well as thermal denaturation studies. Furthermore, the structure of this novel crosslinked duplex is compared with that of less-distorting mitomycin ICLs using Molecular Dynamics simulations.

Microbial hydrolytic defluorinases and dechlorinases assayed by coupling the reaction product to NAD-linked dehydrogenases will facilitate screening for and characterising new and known enzymes.

ABSTRACT

Many environmental pollutants have a fluorine or chlorine atom on a carbon atom adjacent to a carboxylic acid. These α-halocarboxylic acids include heavily regulated compounds such as per- and polyfluorinated substances (PFAS). Due to PFAS persistence in the environment, there is intense interest in characterising the biodegradation of α-halocarboxylic acids. Their initial biodegradation often proceeds via defluorinase enzymes that catalyse hydrolytic removal of alpha fluorine or chlorine atoms. These enzymes can dehalogenate both mono-halocarboxylate and dihalocarboxylate substrates, generating α-hydroxy and α-ketocarboxylic acid products, respectively. To enable continuous monitoring of defluorinase activity, we identified, purified and optimised dehydrogenases from Limosilactobacillus fermentum JN248 and Enterococcus faecium IAM10071 that reacted with the specific α-hydroxy and α-ketocarboxylic acid products of the defluorinases. The dehydrogenases make or consume NADH, measured by absorbance readings at 340 nm, thus allowing continuous measurement of defluorinase activity using a spectrophotometer. Using the coupled assay, purified defluorinases from a Delftia sp. and a Dechloromonas sp. were compared with respect to substrate specificity. The Delftia defluorinase demonstrated superior activity with most substrates, including difluoroacetate. To our knowledge, this is the first report of a coupled-enzyme continuous assay method for enzymes that catalyse the hydrolysis of α-halocarboxylic acids.

Copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that utilizes GGPP as a substrate to generate the bicyclic diterpene copalyl diphosphate. The various stereoisomers of copalyl diphosphate establish the greater family of labdane natural products, many of which have environmental and medicinal impact. Understanding structure-function relationships in class II diterpene synthases is crucial for guiding protein engineering campaigns aimed at the generation of diverse bicyclic diterpene scaffolds. However, only a limited number of structures are available for class II cyclases from bacteria, plants, and humans, and no structures are available for a class II cyclase from a fungus. Further, bifunctional class II terpene synthases have not been investigated with regard to substrate channeling between the prenyltransferase and the cyclase. Here, we report the 2.9 [A]-resolution cryo-EM structure of the 63-kD class II cyclase domain from PvCPS. Comparisons with bacterial and plant copalyl diphosphate synthases reveal conserved residues that likely guide the formation of the bicyclic labdane core, but divergent catalytic dyads that mediate the final deprotonation step of catalysis. Substrate competition experiments reveal preferential GGPP transit from the PvCPS prenyltransferase to the cyclase, even when prepared as separate constructs. These results are consistent with a model in which transient prenyltransferase-cyclase association facilitates substrate channeling due to active site proximity. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/671325v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@7e8233org.highwire.dtl.DTLVardef@196087aorg.highwire.dtl.DTLVardef@1069e18org.highwire.dtl.DTLVardef@1756858_HPS_FORMAT_FIGEXP M_FIG C_FIG

The nickel pincer nucleotide (NPN) cofactor catalyzes the racemization/epimerization of -hydroxy acids in enzymes of the LarA family. The established proton-coupled hydride transfer mechanism requires two catalytic histidine residues that alternately act as general acids and general bases. Notably, however, a fraction of LarA homologs (LarAHs) lack one of the active site histidine residues, replacing it with an asparaginyl side chain that cannot participate in acid/base catalysis. Here, we investigated two such LarAHs and solved their cryo-electron microscopic structures with and without loaded NPN cofactor, respectively. The structures revealed a consistent octameric assembly that is unprecedented in the LarA family and unveiled a new set of active site residues that likely recognize and process substrates differently from those of the well-studied LarAHs. Genomic context analysis suggested their potential involvement in carbohydrate metabolism. Together, these findings lay the groundwork for expanding the breadth of reactions and the range of mechanisms of LarA enzymes.

Applied and Environmental Microbiology, Volume 91, Issue 9, September 2025.

Nature Catalysis, Published online: 22 August 2025; doi:10.1038/s41929-025-01386-7

The radical S-adenosylmethionine (SAM) enzyme, AbmM, catalyses a replacement of the ring oxygen of a sugar with sulfur. However, how this reaction takes place is unknown. Now, an [Fe4S4] cluster is shown to have a dual role in catalysis. It functions in the reductive cleavage of SAM and is the donor of the appended sulfur atom.

In this work a computational study shows the differences in binding and catalysis of a nonheme iron oxygenase with tethered versus nontethered substrate. The tethered substrate is positioned in an ideal orientation near the active site, while the nontethered substrate shows more dynamics and is not positioned well for double bond epoxidation.

Abstract

Enzymes usually react with a free substrate, although some examples have appeared in the literature of enzymes that utilize a substrate tethered to a protein carrier. However, it is not clear what advantage the tethering has, and therefore a computational study was performed. In particular, we report here the first computational study on the nonheme iron dioxygenase involved in the epoxidation reaction during the dapdiamide biosynthesis (DdaC) and investigate tethered and nontethered substrates. Molecular dynamics (MD) simulations show that the protein carrier applies pressure onto the surface of the protein and influences the fold and active site description and leads to differences in substrate-oxidant interactions. Quantum chemical calculations give much lower epoxidation barriers for the activation of the tethered substrate by 7 kcal mol⁻¹ over the nontethered substrate and highlight the advantage of a tethered substrate in catalysis.

Nature Synthesis, Published online: 19 August 2025; doi:10.1038/s44160-025-00866-9

Combining hydrogen-atom transfer for prochiral radical formation, organic-dye-modulated single-electron transfer and an engineered thiamine-dependent enzyme, a photobiocatalytic platform is developed for assembling C(sp2)–C(sp3) bonds via benzylic C(sp3)–H and aldehyde C(sp2)–H oxidative cross-coupling under mild conditions.

A novel CO₂-fixing enzyme, phosphoenolpyruvate carboxylase from Cannabis sativa, shows 146.8 U mg^{– 1} activity. Protein engineering improves its activity twofold and stability 31-fold, yielding a total turnover number of 1.44 × 105 mol mol^{– 1}, making it a highly efficient CO₂ fixation catalyst.

The extraction of excess CO₂ from the environment requires favorable carbon sequestration methods, among which biochar sequestration is one of the most effective approaches. Therefore, to meet the growing demand for high-efficiency carbon fixation and enhanced metabolic flux in biological carbon sequestration, the development of high-performance phosphoenolpyruvate carboxylase (PEPC) is of significant importance. In this study, a high-activity PEPC from Cannabis sativa (CsPEPC) is identified. However, the enzyme exhibits poor thermal stability, with a half-life of only 13 min at 50 °C. To improve CsPEPC performance, diverse strategies, including molecular dynamics, FoldX energy calculations, and PROSS-based prediction, are integrated to screen beneficial variants. The resulting mutant CsPEPC^C886R shows a twofold increase in activity (249.2 U mg^{– 1} at 50 °C) and fivefold greater stability. Furthermore, the addition of 25% glycerol as a protein stabilizer significantly extends the half-life of CsPEPC^C886R by 31-fold, ultimately increasing the half-life to 2178 min. In vitro production experiments show that the optimal mutant CsPEPC^C886R generates 116.8 mM oxaloacetate within 270 min. This work provides a promising PEPC variant for biological CO₂ fixation, which holds great potential for improving carbon assimilation and supports the development of efficient, carbon-neutral biosynthetic pathways.

Enzymes may not only generate catalytic competent conformations for enzyme-substrate complexes but also be able to preserve the reactive conformations to the stage when the reactions reach TS to allow catalytic residues to exert their effects. Such property could be lost for some mutants, leading to diminished TS stabilization and significant reduction of catalytic efficiency. Blue: enzyme; Yellow: substrate; Orange: the altered structure of substrate in TS.

Abstract

Determining strategies used by enzymes to achieve enormous rate enhancements has been a central goal in biochemical research. The abilities to form catalytic competent reactive conformations for enzyme-substrate complexes with well-positioned residues and to lower activation barriers through transition state (TS) stabilization are two accepted mechanisms. Enzymes might have evolved to possess other unrecognized strategies to achieve rate enhancements, and identification of such strategies is of fundamental importance. Here we perform a quantum mechanical/molecular mechanical study of carnosine N-methyltransferase 1 and its mutants, and results indicate that enzymes may possess the ability to preserve reactive conformations and prevent their premature distortions into less active forms during reactions. For wild-type, the reactive conformation can be formed in the enzyme-substrate complex and be preserved to the stage when the reaction reaches TS. For Tyr386Ala, Tyr396Asp, and Tyr398Ala, the wild-type-like reactive conformations can be formed in the mutant-substrate complexes but undergo distortions into less active conformations in the early stage of the reaction before TS. Such premature distortions of the wild-type-like reactive conformations in the mutant complexes prevent residues from fully exerting catalytic effects. Thus, the ability to preserve reactive conformations may be an important feature for some enzymes and can make important contributions to catalytic efficiency.

BorF is a short-chain flavin reductase from a desert soil bacterium that uses NADH to reduce FAD to FADH2, which is used by the tryptophan-6-halogenase BorH to chlorinate tryptophan in the biosynthetic pathway of borregomycin A. The X-ray crystal structure of BorF bound to FAD was solved to 2.37 [A] by molecular replacement and consists of a homodimer of single-domain protomers with a Greek key split {beta}-barrel topology containing a domain-swapped N-terminal -helix, as seen in other members of this family. Insertions and deletions in the region between 3 and {beta}5 lead to a variety of different conformations of the adenosine portion of FAD bound to BorF and structurally related reductases. Comparison of the FAD-bound structures of BorF and BorH suggests that FAD must completely dissociate from BorH in order to be reduced by BorF.

Photoenzymes enable light-driven chemistry inside the chiral environment of a protein. In their Communication (e202505431), Cathleen Zeymer et al. show that natural sugar dehydrogenases utilizing the redox cofactor pyrroloquinoline quinone (PQQ, in orange) can be repurposed for enantioselective photoredox catalysis. Upon blue-light irradiation, radical cyclizations are catalyzed. This work adds a new class of enzymes to the toolbox of photobiocatalysis. Cover art designed by Benjamin Large (Sc·EYE·nce).