Heme enzymes can perform a wide range of reactions in biological systems, often controlled by the heme surrounding amino acids or in conjunction with redox partners. Recently, we resolved the cryo-EM structure of styrene oxide isomerase, a transmembrane protein that catalyzes the isomerization of epoxides into carbonyl compounds. We discovered that heme acts as the cofactor and catalytic center, with tyrosine serving as the key residue in catalysis. While it is evident that tyrosine coordinates the substrate in the active site, the catalytic mechanism is not fully established yet. In this work, we advanced the investigation into a homologous enzyme to explore whether tyrosine plays a conserved role in catalysis. Site-directed mutagenesis of this tyrosine (Y131) to Ala, His, Ser, and Phe demonstrated a significant effect on the activity and kinetic parameters. The pH-dependent activity assay and inhibition of the heme site with carbon monoxide illustrated both ferric heme and tyrosine to be crucial for catalysis. The NMR-based enzyme kinetics suggested that heme b acts as a Lewis-acid in ring opening and Y131 facilitates the trans-methyl/hydride shift to perform the Meinwald-rearrangement reaction. Furthermore, our findings indicate that active heme can be utilized for various reactions, functioning as peroxidase with a turnover number (k_cat) of 2 s^–1and as a peroxygenase with a total yield of 10% phenylacetaldehyde. Although its peroxidase activity is ca. 1000-fold less efficient compared to dye decolorizing peroxidase DyP, the multifunctionality of ZcSOI makes it an intriguing enzyme for application studies. This work deepens our understanding of the SOI mechanism and also reports on the less efficient peroxidase and peroxygenase activity. This, in turn makes SOI a promising candidate for protein engineering to apply in the field of biotechnology, such as improving the epoxidation and isomerization of styrene to produce phenylacetaldehyde by a single enzyme.

The mitochondrial outer membrane iron–sulphur ([Fe-S]) protein mitoNEET has been extensively studied as a target of the anti-inflammatory and type-2 diabetes drug pioglitazone and as a protein affecting mitochondrial respiratory rate. Despite these extensive past studies, its molecular function has yet to be discovered. Here, we applied an interdisciplinary approach and discovered an explicit nitric oxide (NO) access site to the mitoNEET [2Fe-2S] cluster. We found that O2 and pioglitazone block NO access to the cluster, suggesting a molecular function for the mitoNEET [2Fe-2S] cluster in mitochondrial signal transduction. Our discovery hints at a new pathway via which mitochondria can sense hypoxia through O2 protection of the mitoNEET [2Fe-2S] cluster, a new paradigm in understanding the importance of [Fe-S] clusters for gasotransmitter signal transduction in eukaryotes.

More than ten ergot alkaloids comprising both natural and semi-synthetic products are used to treat various diseases. The central C ring forms the core pharmacophore for ergot alkaloids, giving them structural similarity to neurotransmitters, thus enabling their modulation of neurotransmitter receptors. The haem catalase chanoclavine synthase (EasC) catalyses the construction of this ring through complex radical oxidative cyclization. Unlike canonical catalases, which catalyse H2O2 disproportionation, EasC and its homologues represent a broader class of catalases that catalyse O2-dependent radical reactions. We have elucidated the structure of EasC by cryo-electron microscopy, revealing a nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding pocket and a haem pocket common to all haem catalases, with a unique homodimeric architecture that is, to our knowledge, previously unobserved. The substrate prechanoclavine unprecedentedly binds in the NADPH-binding pocket, instead of the previously suspected haem-binding pocket, and two pockets were connected by a slender tunnel. Contrary to the established mechanisms, EasC uses superoxide rather than the more generally used transient haem iron–oxygen complexes (such as compounds I, II and III), to mediate substrate transformation through superoxide-mediated cooperative catalysis of the two distant pockets. We propose that this reactive oxygen species mechanism could be widespread in metalloenzyme-catalysed reactions.

Styrene oxide isomerase (SOI) is a part of the styrene degradation enzyme complex, performing the isomerization of toxic intermediate styrene oxide into phenylacetaldehyde. For many years, the enzyme was believed to be cofactor-independent, and hence, the mechanism of this enzyme was proposed to be acid-base catalysis. Recently, the presence of heme was identified and reported in SOI from Pseudomonas sp. VLB120. Alongside, the membrane localization was also postulated since its discovery but lacks experimental proof. In this study, we highlight the localization of SOIs from two bacterial strains, Rhodococcus opacus 1CP and Zavarzinia compransoris Z-1155, heterologously overproduced in the cell membrane of E. coli via sfGFP-tagged fusions. In addition, the site-directed mutagenesis of acidic and basic amino acids in SOI from 1CP also showcased that histidine-57 is the axial ligand to the heme. Electron paramagnetic resonance (EPR) and biocatalytic assays showed arginine-111 possibly coordinating the propionate group of heme. The functional assays of differently tagged sfGFP with and without linkers, and the truncation of the terminal extension of SOI from 1CP and Z-1155, indicate their possible role in proper substrate channeling. It also supports the previously proposed SOI role as a membrane anchor for other enzymes in styrene degradation pathway. IMPORTANCE: Styrene oxide isomerase (SOI) catalyzes the isomerization of styrene oxide into phenylacetaldehyde in the side chain oxygenation of the styrene degradation pathway. Despite performing a key role in this pathway, the biology and biochemistry of this enzyme are poorly understood, particularly its catalytic mechanism, membrane localization, and structure-function relationships. SOIs from Rhodococcus opacus and Zavarzinia compransoris were produced as SUMO/sfGFP/mCherry fusion proteins. We successfully achieved the overproduction and performed site-directed mutagenesis to understand the catalytic mechanism, performed whole-cell assays, used fluorescent microscopy to assess the membrane localization, and constructed terminal truncations to assess structure-function relationships. The site-directed mutagenesis revealed histidine-57 as the axial ligand for heme. The fluorescence microscopy of sfGFP-fusion showed that SOI is a membrane-bound protein with both termini localized in the cytosol. The difference in activity of differently tagged SOI and truncation of the terminal extension showed that the termini might facilitate proper substrate channelling.

The transition metals tungsten and molybdenum are the heaviest metals found in biological systems and are embedded in the cofactor of several metalloenzymes. As a result of their redox activity, they provide great catalytic power in these enzymes and facilitate chemical reactions that would not occur using only the functionalities of natural amino acids. For their functionality these enzymes depend on a metal cofactor, which consists of at least one metal binding pterin (MPT) and a tungsten or molybdenum ion, but the complete make-up of the cofactor differs per enzyme group. One of these enzyme groups comprises the AOR-family enzymes. These enzymes have the ability to oxidize a range of aldehyde substrates into their corresponding carboxylic acid products. Next to this, they are also the only known catalysts able to perform the thermodynamically challenging reduction reaction of carboxylic acids to aldehydes. These enzymes are currently obtained by purification from the hyperthermophilic archaeon Pyrococcus furiosus. This process, however, does not yield a large amount of enzyme, since it is naturally expressed at moderate levels. For that reason, other production methods need to be considered if the enzyme is to be used on a large scale. These alternatives include the use of a recombinant expression system. The recombinant expression of W-dependent enzymes in different host organisms, such as Escherichia coli, has already been attempted for different enzymes, but with varying success. This shows that more research on the production, and especially incorporation of the metal cofactor, is necessary to achieve a successful production and use of recombinant AOR-family enzymes.

Oleate hydratases open a biocatalytic access to hydroxy fatty acids by hydration of unsaturated fatty acids. Their practical applicability, however, is hampered by their low stability. In this study we report the immobilization of the oleate hydratase from Rhodococcus erythropolis PR4 on functionalized porous, spherical polymer beads. Different carrier materials promoting covalent, hydrophobic, ionic and his-tag affinity were screened and immobilization yields typically >95 % were observed. The highest activity recovery of 32 % was achieved by immobilization via ionic interaction with quaternary ammonium functionalized beads. Biochemical properties of the enzyme immobilized via ionic interaction remain unchanged upon immobilization. The immobilized enzyme was applied for synthesis of 10-hydroxystearic acid remaining stable under process conditions. Conversion of up to 100 mM oleic acid gave 10-hydroxystearic acid achieving a TON of up to 19,000. Successful recycling of the biocatalyst for up to ten cycles further demonstrate its potential for the synthesis of 10-hydroxystearic acid.

The controlled release of drugs using local ionizing radiation presents a promising approach for targeted cancer treatment, particularly when applied in concurrent radio-chemotherapy. In these approaches, radiation-generated reactive species often play an important role. However, the reactive species that can be used to trigger release have low yield and lack selectivity. Here, we demonstrate the generation of highly oxidative species when aqueous solutions containing low concentrations of organochlorides (such as chloroform) are irradiated with ionizing radiation at therapeutically relevant doses. These reactive species were identified as peroxyl radicals, which formed in a reaction cascade between organochlorides and aqueous electrons. We employed stilbene-based probes to investigate the oxidation process, showing double bond oxidation and cleavage. To translate this reactivity into a radiation-sensitive material, we synthesized a micelle-forming amphiphilic block copolymer that has stilbene as the linker between two blocks. Upon exposure to ionizing radiation, the oxidation of stilbene led to the cleavage of the polymer, which induces the dissociation of the block-copolymer micelles and the release of loaded drugs.

Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C–C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R-selectivity of 83% ee. The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R-selectivity >99.9% ee. Michaelis–Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover kcat of 0.71 ± 0.03 s–1 and a Km of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate.

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement—a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound—and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions. (Figure presented.).

Multifunctional, biocompatible magnetic materials, such as iron oxide nanoparticles (IONPs), hold great potential for biomedical applications including diagnostics (e.g., MRI) and cancer therapy. In particular, they can play a crucial role in advancing cancer thermotherapy by generating heat when administered intratumorally and when exposed to an alternating magnetic field. This heat application is often combined with radio- (chemo)therapy and/or imaging. Consequently, the design of materials for such a multimodal approach requires hybrid nanoparticles that retain their magnetic properties while integrating additional functionalities. This work introduces synthesis and investigation of magnetically enhanced nanoparticles with a palladium core (envisioned for future radiolabeling with therapeutic 103Pd) and a magnetic iron oxide shell containing paramagnetic manganese (Pd/Fe|(nMn)-oxide, n = 0.25 and 0.5). Doping the iron oxide lattice with Mn significantly increases magnetic saturation, boosting specific loss power up to 1.7 times compared to that of undoped analogs. Interestingly, higher Mn-content in Pd/Fe|(0.5Mn)-oxide leads to a pronounced Mn outer rim, enhancing the heating efficiency at 346 kHz and 23 mT and contributing to the water exchange on the surface of the paramagnetically doped nanoparticles, resulting in additional T1 MRI contrast. The enhanced magnetic properties of the hybrid Pd/Fe|Mn-oxide nanoparticles enable effective therapeutic outcomes with injection of only small quantities of the material, offering great potential for effective cancer treatment strategies that combine hyperthermia/thermal ablation with radiotherapy while allowing for real-time monitoring via MRI.

This study presents a three-step one pot enzymatic cascade for the synthesis of a δ-lactone. Utilising acetaldehyde, combining 2-deoxyribose-5-phosphate aldolase (DERA) with an alcohol dehydrogenase (ADH) and a cofactor regeneration system this δ-lactone is synthesised with the same stereochemistry as the statin side chain precursor. The initial stage in this cascade involves the double aldol reaction, catalysed by DERA to produce the chiral lactone precursor from the achiral substrate acetaldehyde. The main challenge at this stage is the instability of DERA in the presence of high acetaldehyde concentrations. Therefore, Lactobacillus brevis DERA with a high natural acetaldehyde tolerance was genetically engineered to further improve this property. LbDERA C42M E78K exhibited improved activity and stability (no activity loss over 2 h) compared to the wild type (20% activity loss). In the second stage of the cascade, the aldol product is selectively oxidised to the lactone. A commercially available ADH was identified to selectively catalyse this oxidation using NADP⁺ as electron acceptor. NADP⁺ regeneration was achieved using O₂ as substrate in two different ways: using either photo-activated flavin or NADPH oxidase (NOX). The lactone was successfully purified from the enzymatic cascades from a preparative scale reaction in 97% purity with an optical rotation [α]_D = +34.2° (c = 0.7), proving the feasibility in a multi-enzyme three-step one-pot cascade.

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived.

The Birch reduction is a widely used synthetic tool to reduce arenes to 1,4-cyclohexadienes. Its harsh cryogenic reaction conditions and the dependence on alkali metals have motivated researchers to explore alternative approaches. In anaerobic aromatic compound degrading microbes, class II benzoyl-coenzyme A (CoA) reductases (BCRs) reduce benzoyl-CoA to the conjugated cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA) at a tungsten-bis-metallopterin (MPT) cofactor. Though previous structure-based computational studies were in favor of a Birch-like reduction via W(V)/radical intermediates, any experimental evidence for such a mechanism was lacking. Here, we combined freeze-quench and equilibrium electron paramagnetic resonance (EPR) spectroscopic analyses in H2O, D2O, and H217O with redox titrations using wild-type and molecular variants of the catalytic BamB subunit of class II BCR from the anaerobic bacterium Geobacter metallireducens. We provide spectroscopic evidence for a kinetically competent radical/W(V)-OH intermediate obtained after hydrogen atom transfer from the W-aqua-ligand to the aromatic ring and for an invariant histidine as a proton donor assisting the second electron transfer. Quantum mechanical/molecular mechanical calculations suggest that the unique tetrahydro state of both pyranopterins is essential for the reversibility of enzymatic Birch reduction. This work elucidates nature's solution for the chemically demanding Birch reduction and demonstrates how the reactivity of MPT cofactors can be expanded to highly challenging radical chemistry at the negative limit of the biological redox window.

S-Adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) are highly chemoselective enzymes grouped in C-, N-, O-, S- and halide MTs, depending on the (hetero) atom that acts as the methyl group acceptor. So far, OMTs present the largest group, including many well investigated candidates. The catechol OMT from mammals such as from Rattus norvegicus (RnCOMT) is involved in the metabolism of neurotransmitters like dopamine. It is known to methylate the hydroxyl of the catechol ring in the 3 position. There are also reports showing that the regioselectivity of different COMTs can vary leading to different products with methyl groups in the 3 and or 4 positions. Nevertheless, there was only O-methylation reported for COMTs. Another related MT, the caffeate OMT involved in the lignin biosynthesis of plants has also been reported as a chemoselective enzyme. In nature, S-methylation is a rare phenomenon with different methyl donors being involved in the methyl transfer onto sulfur atoms. Several SAM-dependent MTs are identified as S-methyltransferases (SMTs), these are involved in salvaging pathways and xenobiotic metabolism of cells. Here, we report a new function of three OMTs; RnCOMT, a COMT from Myxococcus xanthus (MxSafC), and a CaOMT from Prunus persica (PpCaOMT) with acceptance towards different aromatic thiol substrates with up to full conversion.

Granulicella tundricola hydroxynitrile lyase (GtHNL) is a manganese dependent cupin that catalyzes the enantioselective synthesis of cyanohydrins. The analysis of its active site shows high similarity with the active site of cupin Tm1459 from Thermotoga maritima, an enzyme that catalyzes the oxidative cleavage of styrene derivatives. GtHNL (GtHNL-WT) was found to catalyze the oxidative cleavage of α-methyl styrene, too. The conversion of α-methyl styrene yielded 23.6 ± 0.8% of acetophenone after 20 h. On the other hand, Tm1459 was not able to catalyze the synthesis of cyanohydrins efficiently. A low yield of rac-mandelonitrile was obtained from benzaldehyde and HCN using either Tm1459-WT or Tm1459-C106L, a variant more active in oxidative catalysis. On the basis of the molecular analysis of GtHNL and Tm1459 active sites, the variants GtHNL-H96A, GtHNL-H96F, and GtHNL-A40H/V42T/H96A/Q110H were produced and evaluated for improved catalytic activity toward oxidative cleavage of styrenes. The amino acid substitution H96A liberates an additional manganese coordination position and enlarges the GtHNL-WT active site cavity. Similarly, the amino acid substitution H96F liberates a coordination site as described for the GtHNL-H96A variant but without enlarging the active site space. All variants were able to catalyze the oxidative cleavage of styrene derivatives. The best results were observed using GtHNL-H96A as catalyst. It displayed a higher yield of acetophenone (42%) as compared to GtHNL-A40H/V42T/H96A/Q110H (12%) and GtHNL-H96F (11%) after 20 h of reaction time. No oxidation of Mn(II) to Mn(III) could be detected by electron paramagnetic resonance (EPR), whereas evidence for a radical mechanism is presented. Control reactions using 0.1 and 0.5 mM of MnCl₂ in the absence of enzyme showed no significant oxidation reaction.

Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5–10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 g_product·L^-1·h⁻¹·mg_protein^-1, for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN₃ solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides.

In this review the current state-of-the-art of S-adenosylmethionine (SAM)-dependent methyltransferases and SAM are evaluated. Their structural classification and diversity is introduced and key mechanistic aspects presented which are then detailed further. Then, catalytic SAM as a target for drugs, and approaches to utilise SAM as a cofactor in synthesis are introduced with different supply and regeneration approaches evaluated. The use of SAM analogues are also described. Finally O-, N-, C- and S-MTs, their synthetic applications and potential for compound diversification is given.

A virus hijacks host cellular machineries and metabolites in order to reproduce. In response, the innate immune system activates different processes to fight back. Although many aspects of these processes have been well investigated, the key roles played by iron–sulfur [FeS] clusters, which are among the oldest classes of bio-inorganic cofactors, have barely been considered. Here we discuss how several [FeS] cluster-containing proteins activate, support and modulate the innate immune response to restrict viral infections, and how some of these proteins simultaneously support the replication of viruses. We also propose models of function of some proteins in the innate immune response and argue that [FeS] clusters in many of these proteins act as biological ‘fuses’ to control the response. We hope this overview helps to inspire future research in the emerging field of bio-inorganic virology/immunology and that such studies may reveal new molecular insight into the links between viral infections and diseases like cancer and neurodegeneration. [Figure not available: see fulltext.]

We used a series of modified/substituted GGH analogues to investigate the kinetics of Cu(ii) binding to ACTUN peptides. Rules for rate modulation by 1^st and 2^nd sphere interactions were established, providing crucial insight into elucidation of the reaction mechanism and its contribution to biological copper transport.