Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

Construction of a dual-signal readout platform for effective glutathione S-transferase sensing based on polyethyleneimine-capped silver nanoclusters and cobalt-manganese oxide nanosheets with oxidase-mimicking activity.

A novel dual-mode fluorometric and colorimetric sensing platform is reported for determining glutathione S-transferase (GST) by utilizing polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) and cobalt-manganese oxide nanosheets (CoMn-ONSs) with oxidase-like activity. Abundant active oxygen species (O2•-) can be produced through the CoMn-ONSs interacting with dissolved oxygen. Afterward, the pink oxDPD was generated through the oxidation of colorless N,N-diethyl-p-phenylenediamine (DPD) by O2•-, and two absorption peaks at 510 and 551 nm could be observed. Simultaneously, oxDPD could quench the fluorescence of PEI-AgNCs at 504 nm via the inner filter effect (IFE). However, in the presence of glutathione (GSH), GSH prevents the oxidation of DPD due to the reducibility of GSH, leading to the absorbance decrease at 510 and 551 nm. Furthermore, the fluorescence at 504 nm was restored due to the quenching effect of oxDPD on decreased PEI-AgNCs. Under the catalysis of GST, GSH and1-chloro-2,4-dinitrobenzo (CDNB) conjugate to generate an adduct, initiating the occurrence of the oxidation of the chromogenic substrate DPD, thereby inducing a distinct colorimetric response again and the significant quenching of PEI-AgNCs. The detection limits for GST determination were 0.04 and 0.21 U/L for fluorometric and colorimetric modes, respectively. The sensing platform illustrated reliable applicability in detecting GST in real samples.

Temperature-resilient and sustainable Mn-MOF oxidase-like nanozyme (UoZ-4) for total antioxidant capacity sensing in some citrus fruits: Breaking the temperature barrier.

Current nanozyme applications rely heavily on peroxidase-like nanozymes and are limited to a specific temperature range, despite notable advancements in nanozyme development. In this work, we designed novel Mn-based metal organic frameworks (UoZ-4), with excellent oxidase mimic activity towards common substrates. UoZ-4 showed excellent oxidase-like activity (with Km 0.072 mM) in a wide range of temperature, from 10 °C to 100 °C with almost no activity loss, making it a very strong candidate for psychrophilic and thermophilic applications. Ascorbic acid, cysteine, and glutathione could quench the appearance of the blue color of oxTMB, led us to design a visual-based sensing platform for detection of total antioxidant capacity (TAC) in cold, mild and hot conditions. The visual mode successfully assessed TAC in citrus fruits with satisfactory recovery and precisions. Cold/hot adapted and magnetic property will broaden the horizon of nanozyme applications and breaks the notion of the temperature limitation of enzymes.

Enhanced oxidase mimic activity of raspberry-like N-doped Mn3O4 with oxygen vacancies for efficient colorimetric detection of gallic acid coupled with smartphone.

The content of gallic acid (GA) is positively correlated with the quality grade of tea. Here, we developed a colorimetric method based on raspberry-like N-doped Mn3O4 nanospheres (N-Mn3O4 NSs) with oxidase-like activity for GA assay. Modulating the electronic structure of Mn3O4 by N doping could promote the catalysis ability, and the produced oxygen vacancies (OVs) can provide high surface energy and abundant active sites. The N-Mn3O4 NSs presented low Michaelis-Menten constant (Km) of 0.142 mM and maximum initial velocity (Vmax) of 9.8 × 10-6 M s-1. The sensor exhibited excellent analytical performance towards GA detection, including low LOD (0.028 µM) and promising linear range (5 âˆ¼ 30 µM). It is attributed that OVs and O2- participated in TMB oxidation. Based on the reaction color changes, a visualized semi-quantitative GA detection could be realized via a smartphone-based system. It could be applied for evaluating GA quality in market-purchased black tea and green tea.

G-quadruplex DNA-based colorimetric biosensor for the ultrasensitive visual detection of strontium ions using MnO2 nanorods as oxidase mimetics.

Strontium-90 (90Sr) is a major radioactive component that has attracted great attention, but its detection remains challenging since there are no specific energy rays indicative of its presence. Herein, a biosensor that is capable of rapidly detecting Sr2+ ions is demonstrated. Simple colorimetric method for sensitive detection of Sr2+ with the help of single-stranded DNA was developed by preparing MnO2 nanorods as oxidase mimic catalysis 3,3',5,5'-tetramethylbenzidine (TMB). Under weakly acidic conditions, MnO2 exhibited a strong oxidase-mimicking activity to oxidize colorless TMB into blue oxidation products (oxTMB) with discernible absorbance signals. Nevertheless, the introduction of a guanine-rich DNA aptamer inhibited MnO2-mediated TMB oxidation and reduced oxTMB formation, resulting in blue fading and diminished absorbance. Upon the addition of strontium ions to the system, the aptamers formed a stable G-quadruplex structure with strontium ions, thereby restoring the oxidase-mimicking activity of MnO2. Under the best experimental conditions, the absorbance exhibits a linear relationship with the Sr2+ concentration within the range 0.01-200 µM, with a limit of detection of 0.0028 µM. When the concentration of Sr2+ from 10-8 to 10-6 mol L-1, a distinct color change gradient could be observed in paper-based sensor. We successfully applied this approach to determine Sr2+ in natural water samples, obtaining recoveries ranging from 97.6 to 103% with a relative standard deviation of less than 5%. By providing technical solutions for detection, our work contributed to the effective monitoring of transportation of radioactive Sr in the environment.

Reduction of molecular oxygen in flavodiiron proteins - Catalytic mechanism and comparison to heme-copper oxidases.

The family of flavodiiron proteins (FDPs) plays an important role in the scavenging and detoxification of both molecular oxygen and nitric oxide. Using electrons from a flavin mononucleotide cofactor molecular oxygen is reduced to water and nitric oxide is reduced to nitrous oxide and water. While the mechanism for NO reduction in FDPs has been studied extensively, there is very little information available about O2 reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O2 reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O2 molecule has bound to the diferrous diiron active site and before the OO bond can be cleaved. This is in contrast to the mechanism for NO reduction, where both NN bond formation and NO bond cleavage occurs from the same starting structure without any further reduction, according to both experimental and computational results. This computational result for the O2 reduction mechanism should be possible to evaluate experimentally. Another difference between the two substrates is that the actual OO bond cleavage barrier is low, and not involved in rate-limiting the reduction process, while the barrier connected with bond cleavage/formation in the NO reduction process is of similar height as the rate-limiting steps. We suggest that these results may be part of the explanation for the generally higher activity for O2 reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O2 reduction reaction in the family of heme­copper oxidases.

Engineering the activity and thermostability of a carboxylic acid reductase in the conversion of vanillic acid to vanillin.

Vanillin is a valuable natural product that can be used as a fragrance and additive. Recent research in the biosynthesis of vanillin has brought attention to a key enzyme, carboxylic acid reductase (CAR), which catalyzes the reduction of vanillic acid to vanillin. Nevertheless, the biosynthesis of vanillin is hampered by the low activity and stability of CAR. As such, a rational design campaign was conducted on a well-documented carboxylic acid reductase from Segniliparus rugosus (SrCAR), using vanillic acid as the model substrate. After combined active site saturation and iterative site-specific mutagenesis, the best quadruple mutant N292H/K524S/A627L/E1121W (M3) was successfully obtained. In comparison to the wildtype SrCAR, M3 demonstrated a 4.2-fold increase in catalytic efficiency (kcat/Km), and its half-life (t1/2) was enhanced by 3.8 times up to 385.08 minutes at 40 °C. In silico docking and molecular dynamics simulation provided insights into the improved activity and stability. In the subsequent preparative-scale reaction with 100 mM (16.8 g L-1) vanillic acid, the whole cell catalysis utilizing M3 produced 10.15 g·L-1 of vanillin and 1.11 g·L-1 of vanillyl alcohol, respectively. This work demonstrates a dual improvement in the activity and thermal stability of SrCAR, thereby potentially facilitating the application of carboxylic acid reductase in the biosynthesis of vanillin.

CoO/Co-graphene quantum dots as an oxidative mimetic nanozyme for the colorimetric detection of L-cysteine.

The preparation of cobalt-based nanozymes with high oxidase-like activity still needs more efforts. In this paper, we report the synthesis of a CoO/Co-tryptophan-functional graphene quantum dot hybrid (CoO/Co-Try-GQD). Firstly, cobalt ions coordinate with the indole nitrogen on Try-GQD to form a complex, followed by thermal reduction and oxidation. The resulting hybrid presents a three-dimensional network structure, and CoO/Co nanoparticles are uniformly dispersed on the graphene sheet with an average size of 10 ± 0.24 nm. This unique structure improved the oxidase-like activity of the hybrid, enabling it to catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to rapidly produce deep blue ox-TMB with a strong absorbance at 652 nm (A652). A colorimetric method was developed for the highly sensitive determination of L-cysteine (L-cys) based on the inhibition of the hybrid's oxidase-like activity and low A652 caused by the binding of L-cys with Co atoms on CoO/Co via the Co-S bond. The A652 linearly decreased with increasing L-cys concentration in the range of 0.05-2 µM, and the detection limit was 0.032 µM. Further, the established method has been successfully applied to the determination of L-cys in milk.

Single-holed cobalt - nitrogen - carbon hollow structure with oxidase-mimicking activity for the chemiluminescence determination of ß - galactosidase activity.

A single-holed cobalt - nitrogen - carbon (Co - N - C) hollow structure nanozyme has been fabricated by in situ growth of zeolitic imidazolate framework (ZIF - 67) on the polystyrene (PS) sphere and following treatment by high-temperature carbonization. The Co - N - C nanostructure mimics the activity of oxidase and can activate O2 into reactive oxygen species (ROS), giving a remarkable enhancement on the chemiluminescence (CL) signal of luminol - O2 reaction. The Co - N - C oxidase mimic has further been exploited in the biosensing field by the determination of the activity of ß - galactosidase (ß - gal). The CL method for ß - gal activity has a linear range of 0.5 mU·L-1 to 5.0 U·L-1, a detection limit of 0.167 mU·L-1, and the precision of 3.1% (5.0 U·L-1, n = 11). This method has been employed to assess inhibitor screening of ß - gal and determine activity of ß - gal in spiked human serum samples.

Regiodivergent Radical Termination for Intermolecular Biocatalytic C-C Bond Formation.

Radical hydrofunctionalizations of electronically unbiased dienes are challenging to render regioselective, because the products are nearly identical in energy. Here, we report two engineered FMN-dependent "ene"-reductases (EREDs) that catalyze regiodivergent hydroalkylations of cyclic and linear dienes. While previous studies focused exclusively on the stereoselectivity of alkene hydroalkylation, this work highlights that EREDs can control the regioselectivity of hydrogen atom transfer, providing a method for selectively preparing constitutional isomers that would be challenging to prepare using traditional synthetic methods. Engineering the ERED from Gluconabacter sp. (GluER) furnished a variant that favors the γ,δ-unsaturated ketone, while an engineered variant from a commercial ERED panel favors the δ,ε-unsaturated ketone. The effect of beneficial mutations has been investigated using substrate docking studies and the mechanism probed by isotope labeling experiments. A variety of α-bromo ketones can be coupled with cyclic and linear dienes. These interesting building blocks can also be further modified to generate difficult-to-access heterocyclic compounds.

Hydrogel-involved portable colorimetric sensor based on oxidase mimic Fe/Co-NC for acetylcholinesterase detection and pesticides inhibition assessment.

Herein, we synthesized a novel N-doped carbon layer encapsulated Fe/Co bimetallic nanoparticles (Fe/Co-NC), which exhibited superior oxidase-like activity due to the facilitation of electron penetration and the formation of metal-nitrogen active sites. Fe/Co-NC could catalyze the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) to blue oxTMB. Acetylcholinesterase (AChE) could catalyze the hydrolysis of thioacetylcholine to produce reducing thiocholine, which prevented TMB from oxidation. Thus, a portable hydrogel colorimetric sensor was developed for on-site and visual monitoring of AChE with the detection limit of 0.36 U L-1, and successfully applied to detect AChE in human erythrocyte samples. Furthermore, this platform was used to investigate the inhibition of triazophos on AChE activity.

Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase.

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Enhanced the intrinsic oxidase-like activity of chiral carbon dots via manganese doping for selective catalytic oxidation.

It is highly desirable to design and construct chemical catalysts with high activity and specificity as the alternatives of natural enzymes for industrial application. Chiral carbon dots (CDs), possessing both the intrinsic enzyme-like activity and specific recognition ability, are one of good candidates for enzyme-like catalysts. However, their catalytic activity is far from that of natural enzymes and needs to be enhanced. In this work, the modulation of the chiral structure and catalytic activity of chiral CDs with intrinsic oxidase-like activity was implemented by manganese (Mn) doping. Under the light condition, chiral CDs (l-Ser-CDs and d-Ser-CDs) derived from chiral serine (Ser) show weak catalytic activity and low selectivity toward the oxidation of L type of dopamine (l-DOPA), whereas the Mn functionalized chiral CDs (l-Mn-CDs or d-Mn-CDs) exhibit 6.9 times higher in catalytic activity and 2.9 times in selectivity ratio (SR) than Ser-CDs. Mn-CDs involve two-path catalytic process, in which the photogenerated electrons could reduce O2 to O2- as the active species and the holes would oxidize DOPA directly. Moreover, doping of Mn enables the CDs to generate more O2-. Besides, l-Mn-CDs have higher catalytic activity than that of d-Mn-CDs (+54.2 %), and the chiral Mn-CDs have stronger selective adsorption capacity towards chiral DOPA than Ser-CDs. Our work provides a new method for designing and preparing novel chiral artificial enzymes.

Unraveling the multiplicity of geranylgeranyl reductases in Archaea: potential roles in saturation of terpenoids.

The enzymology of the key steps in the archaeal phospholipid biosynthetic pathway has been elucidated in recent years. In contrast, the complete biosynthetic pathways for proposed membrane regulators consisting of polyterpenes, such as carotenoids, respiratory quinones, and polyprenols remain unknown. Notably, the multiplicity of geranylgeranyl reductases (GGRs) in archaeal genomes has been correlated with the saturation of polyterpenes. Although GGRs, which are responsible for saturation of the isoprene chains of phospholipids, have been identified and studied in detail, there is little information regarding the structure and function of the paralogs. Here, we discuss the diversity of archaeal membrane-associated polyterpenes which is correlated with the genomic loci, structural and sequence-based analyses of GGR paralogs.

Construction of Mn-decorated zeolitic imidazolate framework-90 nanostructure as superior oxidase-like mimic for colorimetric detection of glucose and choline.

A Mn decorated zeolitic imidazolate framework-90 (ZIF-90) nanozyme (Mn/ZIF-90) was constructed through an effective and rapid post-synthetic strategy for the first time. The Mn in Mn/ZIF-90 exists in mixed valence states, which is doped to the ZIF-90 through the formation of Mn-O bond. The Zn-N coordination structure of ZIF-90 may change the electronic arrangement of oxygen atoms in the free carbonyl groups (-CHO), allowing the coordination of Mn with O. The prepared Mn/ZIF-90 possesses outstanding oxidase-like activity and remarkable stability. Besides, the catalytic activity of Mn/ZIF-90 can be inhibited in the presence of H2O2. Therefore, using the Mn/ZIF-90-triggered chromogenic reaction of 3,3',5,5'-tetramethylbenzidine (TMB) as an amplifier, a versatile enzyme cascade-based colorimetric method for the detection of glucose and choline with good sensitivity and selectivity was developed. The linear ranges for glucose and choline are 6.25-500 µM and 5-1000 µM, respectively. Furthermore, the developed method was applied in the detection of glucose and choline in rabbit plasma samples, and the recoveries are 89.5-107.3 % and 96.0-109.3 %, respectively. In short, the simple and efficient post-synthetic doping method may provide a new thought for the rational designs of enzyme mimics with improved catalytic performance. Moreover, the colorimetric method based on the excellent catalytic activity of Mn/ZIF-90 may be extended to detect other H2O2-generating or consuming molecules and evaluate the activity of bio-enzymes that can catalyze the generation of glucose or choline.

Structure of methyltransferase RedM that forms the dimethylpyrrolinium of the bisindole reductasporine.

Bisindoles are biologically active natural products that arise from the oxidative dimerization of two molecules of l-tryptophan. In bacterial bisindole pathways, a core set of transformations is followed by the action of diverse tailoring enzymes that catalyze reactions that lead to diverse bisindole products. Among bisindoles, reductasporine is distinct due to its dimethylpyrrolinium structure. Its previously reported biosynthetic gene cluster encodes two unique tailoring enzymes, the imine reductase RedE and the dimethyltransferase RedM, which were shown to produce reductasporine from a common bisindole intermediate in recombinant E. coli. To gain more insight into the unique tailoring enzymes in reductasporine assembly, we reconstituted the biosynthetic pathway to reductasporine in vitro and then solved the 1.7 Å resolution structure of RedM. Our work reveals RedM adopts a variety of conformational changes with distinct open and closed conformations, and site-directed mutagenesis alongside sequence analysis identifies important active site residues. Finally, our work sets the stage for understanding how RedM evolved to react with a pyrrolinium scaffold and may enable the development of new dimethyltransferase catalysts.

The energetics and evolution of oxidoreductases in deep time.

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation-reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation-reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.

Development of a rapid assay for ß-etherase activity using a novel chromogenic substrate.

Biocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as ß-etherases, capable of breaking ß-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying ß-etherase activity was developed based on the ß-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate ß-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized. Kintetic monitoring of ρ-nitrophenolate release at 410 nm over 10 min, using recombinant LigF from Sphingobium sp SYK-6, LigF-AB and LigE-AB from Althererytrobacter sp B11, yielded enzimatic activities of 404. 3 mU/mg, 72 mU/mg, and 50 mU/mg, respectively. This method is applicable in a pH range of 7.0-9.0, with a sensitivity of up to 50 ng of enzyme, exhibiting no interference with lipolytic, glycolytic, proteolytic, and oxidoreductase enzymes.

Ultrathin porous PdCu metallenezymes as oxidase mimics for colorimetric analysis.

Ultrathin porous and highly curved two-dimensional PdCu alloy metallene are shown to be highly efficient oxidase mimics. Serving as intrinsic oxidase mimic, the ultrathin porous structure of the PdCu metallenezymes could effectively utilize all the Pd atoms of the metallenezymes during catalytic reactions. By using the oxidation capability of 3,3'5,5'-tetramethylbenzidine as distinctive chromogenic substrate, the PdCu metallenezymes was used as oxidase-like mimics for determination of total antioxidant capacity (TAC) of vitamin C containing real products including fresh orange juice, commercial beverages, Vitamin C tablets and dermo-cosmetic products. AAP was hydrolyzed using ALP to generate AA and the corresponding ALP activity was successfully detected in the 0-100 U/L range with a lowest detection limit of 0.9 U/L. This study demonstrates the significant catalytic performance and oxidase-like activity of PdCu metallene nanozyme providing a strategy to develop a TAC assay for the assessment of antioxidant food quality as well as oxidative stress in skin and health care products.

Bioinspired porous three-coordinated single-atom Fe nanozyme with oxidase-like activity for tumor visual identification via glutathione.

Inspired by structures of natural metalloenzymes, a biomimetic synthetic strategy is developed for scalable synthesis of porous Fe-N3 single atom nanozymes (pFeSAN) using hemoglobin as Fe-source and template. pFeSAN delivers 3.3- and 8791-fold higher oxidase-like activity than Fe-N4 and Fe3O4 nanozymes. The high catalytic performance is attributed to (1) the suppressed aggregation of atomically dispersed Fe; (2) facilitated mass transfer and maximized exposure of active sites for the created mesopores by thermal removal of hemoglobin (2 ~ 3 nm); and (3) unique electronic configuration of Fe-N3 for the oxygen-to-water oxidation pathway (analogy with natural cytochrome c oxidase). The pFeSAN is successfully demonstrated for the rapid colorimetric detection of glutathione with a low limit of detection (2.4 nM) and wide range (50 nM-1 mM), and further developed as a real-time, facile, rapid (~6 min) and precise visualization analysis methodology of tumors via glutathione level, showing its potentials for diagnostic and clinic applications.