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.@en

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.@en

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.

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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.@en

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.).

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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.

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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.

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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.

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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.

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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.

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Iron deficiency anemia can be treated with oral or intravenous Fe supplementation. Such supplementation has considerable effects on the human microbiome, and on opportunistic pathogenic micro-organisms. Molecular understanding of the control and regulation of Fe availability at the host-microbe interface is crucial to interpreting the side effects of Fe supplementation. Here, we provide a concise overview of the regulation of Fe by the opportunistic pathogen Staphylococcus aureus. Ferric uptake regulator (Fur) plays a central role in controlling Fe uptake, utilization and storage in order to maintain a required value. The micro-organism has a strong preference for heme iron as an Fe source, which is enabled by the Iron-regulated surface determinant (Isd) system. The strategies it employs to overcome Fe restriction imposed by the host include: hijacking host proteins, replacing metal cofactors, and replacing functions by non-metal dependent enzymes. We propose that integrated omics approaches, which include metalloproteomics, are necessary to provide a comprehensive understanding of the metal tug of war at the host-microbe interface down to the molecular level.

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Isothermal titration calorimetry (ITC) is a popular chemical analysis technique that can be used to measure macromolecular interactions and chemical and physical processes. ITC involves the measurement of heat flow to and from a measurement cell after each injection during a titration experiment. ITC has been useful to measure the thermodynamics of macromolecular interactions such as protein-ligand or protein-protein binding affinity and also chemical processes such as enzyme catalyzed reactions. The use of ITC in biocatalysis has a number of advantages as ITC enables the measurement of enzyme kinetic parameters in a direct manner and, in principle, can be used for most enzymes and substrates. ITC approaches have been developed to measure reversible and irreversible enzyme inhibition, the effects of molecular crowding on enzyme activity, the activity of immobilized enzymes and the conversion of complex polymeric substrates. A disadvantage is that in order to obtain accurate kinetic parameters special care has to be taken in proper experimental design and data interpretation, which unfortunately is not always the case in reported studies. Furthermore, special caution is necessary when ITC experiments are performed that include solvents, reducing agents and may have side reactions. An important bottleneck in the use of calorimetry to measure enzyme activity is the relatively low throughput, which may be solved in the future by sensitive chip based microfluidic enzyme calorimetric devices.@en

Granulicella tundricola hydroxynitrile lyase (GtHNL) catalyses the synthesis of chiral (R)‐ cyanohydrins and (R)‐β‐nitro alcohols. The triple variant GtHNL‐A40H/V42T/Q110H (GtHNL‐3V) was immobilised on Celite R‐633 and used in monophasic MTBE saturated with 100 mM KPi buffer pH 7 for the synthesis of (R)‐2‐nitro‐1‐phenylethanol (NPE) in batch and continuous flow systems. Nitromethane was used as a nucleophile. A total of 82% of (R)‐NPE and excellent enantioselectivity (>99%) were achieved in the batch system after 24 hours of reaction time. GtHNL‐3V on Celite R‐ 633 was successfully recycled five times. During more recycling steps a significant decrease in yield was observed while the enantioselectivity remained excellent over eight cycles. The use of a flow system enabled the continuous synthesis of (R)‐NPE. A total of 15% formation of (R)‐NPE was reached using a flow rate of 0.1 mL min⁻¹; unfortunately, the enzyme was not stable, and the yield decreased to 4% after 4 hours on stream. A similar yield was observed during 15 hours at a rate of 0.01 mL min⁻¹. Surprisingly the use of a continuous flow system did not facilitate the process intensification. In fact, the batch system displayed a space‐time‐yield (STY/mgenzyme) of 0.10 g L⁻¹ h⁻¹ mgenzyme⁻¹ whereas the flow system displayed 0.02 and 0.003 g L⁻¹ h⁻¹ mgenzyme⁻¹ at 0.1 and 0.01 mL min⁻¹, respectively. In general, the addition of 1 M nitromethane potentially changed the polarity of the reaction mixture affecting the stability of Celite‐GtHNL‐3V. The nature of the batch system maintained the reaction conditions better than the flow system. The higher yield and productivity observed for the batch system show that it is a superior system for the synthesis of (R)‐NPE compared with the flow approach.

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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.]

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Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M²⁺_R), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M²⁺_A) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M²⁺_A affords the overall lowest energy complex, which drives the transition from M²⁺_R to M²⁺_A. While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures.

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Steady-state kinetics of cytochrome-c dependent denitrifying NO reductases (cNORs) show evidence of substrate inhibition at NO concentrations higher than 10 μM, but the mechanism of inhibition remains unclear. Here, we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to the oxidized cNORs. A differential signal at 1261 cm⁻¹ that downshifts with ¹⁵NO and ¹⁵N¹⁸O is assigned to a ν(NO₂) from a bridging diiron-nitrito complex at the heme-nonheme diron site. Theoretical calculations reproduces observed frequencies and isotope shifts. Our experimental results confirm a prior theoretical study by Blomberg and Siegbahn [Blomberg, M. R., and Siegbahn, P. E. M. Biochemistry 2012, 51, 5173–5186] that proposed substrate inhibition through a radical combination reaction between the diferric μ-oxo group and an NO molecule to form a heme Fe(III)-nitrito-Fe_B(II) inhibitory complex. Stopped-flow experiments suggest that substrate inhibition also occurs after a half-reduction cycle, i.e. when fully-reduced cNOR reduces two NO molecules at the heme-nonheme diferrous active site cluster to produce one N₂O molecule and the diferric cluster. These results support catalytic mechanisms that proceed through isomerization of a diferric-hyponitrite transient complex to produce a bridging diferric μ-oxo group and N₂O without protonation of the putative hyponitrite intermediate.

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Bisphenols are important industrial materials for example for the production of plastics, but are also well known for their adverse health effects, in particular bisphenol A (BPA) is an endocrine disruptor. The widespread use of plastics has raised concerns. Therefore, the removal of bisphenols from wastewater has sparked the interest of the scientific community. This work introduces a novel hybrid technique of micro-nano bubbles assisted laccase (MNB-Lac) to degrade bisphenols in water. The feasibility of MNB-Lac using BPA as a model contaminant was evaluated by comparing with MNB, Lac, ultrasound (UL), UL-Lac, and UL-MNB-Lac. Comprehensive investigations were carried out to understand the specific influences of key process parameters including the initial pollutant concentration, temperature, air intake, pH, outlet pipe length, and Lac concentration on BPA degradation. The alkaline environment and extended length of outlet pipe could improve the degradation efficiency further. MNB-Lac exhibited 2.3–6.2 folds higher BPA degradation and less time than the other above process under the optimal parameters. The mechanism of MNB-Lac revealed that the generation of hydroxyl radical, high O₂ solubility, and high mass transfer efficiency induced by MNB play important roles on enhancing the degradation catalyzed by Lac. MNB-Lac was successfully used for treating bisphenol B, bisphenol C, and the mixture of three bisphenols with high removal efficiency. Subsequently, these degradation products were analyzed by GC–MS. MNB-Lac potentially represents an innovative technology with considerable advantages in contaminant cleanup and time efficiency for treating phenolic contaminated water. Furthermore, the findings provide new insights into the enhancement of the performance of an oxidizing enzyme by introducing MNB technology.

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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.

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Nanoparticles (NPs) have high multifunctional potential to simultaneously enhance implant osseointegration and prevent infections caused by antibiotic-resistant bacteria. Here, we present the first report on using plasma electrolytic oxidation (PEO) to incorporate different combinations of reduced graphene oxide (rGO) and silver (Ag) NPs on additively manufactured geometrically ordered volume-porous titanium implants. The rGO nanosheets were mainly embedded parallel with the PEO surfaces. However, the formation of ‘nano-knife’ structures (particles embedded perpendicularly to the implant surfaces) was also found around the pores of the PEO layers. Enhanced in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus was observed for the rGO+Ag-containing surfaces compared to the PEO surfaces prepared only with AgNPs. This was caused by a significant improvement in the generation of reactive oxygen species, higher levels of Ag+ release, and the presence of rGO ‘nano-knife’ structures. In addition, the implants developed in this study stimulated the metabolic activity and osteogenic differentiation of MC3T3-E1 preosteoblast cells compared to the PEO surfaces without nanoparticles. Therefore, the PEO titanium surfaces incorporating controlled levels of rGO+Ag nanoparticles have high clinical potential as multifunctional surfaces for 3D-printed orthopaedic implants.@en