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1.
Mikrochim Acta ; 191(7): 368, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38833176

ABSTRACT

A colorimetric analysis platform has been successfully developed based on FeCo-NC dual-atom nanozyme (FeCo-NC DAzyme) for the detection of organophosphorus pesticides (OPPs). The FeCo-NC DAzyme exhibited exceptional oxidase-like activity (OXD), enabling the catalysis of colorless TMB to form blue oxidized TMB (oxTMB) without the need for H2O2 involvement. By combining acid phosphatase (ACP) hydrolase with FeCo-NC DAzyme, a "FeCo-NC DAzyme + TMB + ACP + SAP" colorimetric system was constructed, which facilitated the rapid detection of malathion. The chromogenic system was applied to detect malathion using a smartphone-based app and an auxiliary imaging interferogram device for colorimetric measurements, which have a linear range of 0.05-4.0 µM and a limit of detection (LOD) as low as 15 nM in real samples, comparable to UV-Vis and HPLC-DAD detection methods. Overall, these findings present a novel approach for convenient, rapid, and on-site monitoring of OPPs.


Subject(s)
Colorimetry , Limit of Detection , Pesticides , Smartphone , Colorimetry/methods , Pesticides/analysis , Organophosphorus Compounds/analysis , Organophosphorus Compounds/chemistry , Malathion/analysis , Malathion/chemistry , Oxidoreductases/chemistry , Iron/chemistry , Acid Phosphatase/analysis , Acid Phosphatase/chemistry , Benzidines
2.
Protein Sci ; 33(6): e4997, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38723110

ABSTRACT

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3ß3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by ß-ß interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the ß subunit interfaces, with subsequent targeted improvements of the subunits.


Subject(s)
Enzyme Stability , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Models, Molecular , Dioxygenases/chemistry , Dioxygenases/metabolism , Dioxygenases/genetics , Temperature , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Polyethylene Terephthalates/chemistry , Polyethylene Terephthalates/metabolism , Hydrogen-Ion Concentration , Electron Transport Complex III
3.
Nat Commun ; 15(1): 3802, 2024 May 07.
Article in English | MEDLINE | ID: mdl-38714719

ABSTRACT

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.


Subject(s)
Cryoelectron Microscopy , Ferritins , Nuclear Receptor Coactivators , Ferritins/metabolism , Ferritins/chemistry , Ferritins/genetics , Humans , Nuclear Receptor Coactivators/metabolism , Nuclear Receptor Coactivators/chemistry , Nuclear Receptor Coactivators/genetics , Protein Binding , Binding Sites , Iron/metabolism , Autophagy , Models, Molecular , HEK293 Cells , Oxidoreductases/metabolism , Oxidoreductases/chemistry , Oxidoreductases/genetics , Proteolysis , Mutation
4.
Anal Chim Acta ; 1308: 342664, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38740454

ABSTRACT

Nanozymes is a kind of nanomaterials with enzyme catalytic properties. Compared with natural enzymes, nanozymes merge the advantages of both nanomaterials and natural enzymes, which is highly important in applications such as biosensing, clinical diagnosis, and food inspection. In this study, we prepared ß-MnOOH hexagonal nanoflakes with a high oxygen vacancy ratio by utilizing SeO2 as a sacrificial agent. The defect-rich MnOOH hexagonal nanoflakes demonstrated excellent oxidase-like activity, catalyzing the oxidation substrate in the presence of O2, thereby rapidly triggering a color reaction. Consequently, a colorimetric sensing platform was constructed to assess the total antioxidant capacity in commercial beverages. The strategy of introducing defects in situ holds great significance for the synthesis of a series of high-performance metal oxide nanozymes, driving the development of faster and more efficient biosensing and analysis methods.


Subject(s)
Antioxidants , Manganese Compounds , Oxides , Oxides/chemistry , Antioxidants/chemistry , Antioxidants/metabolism , Antioxidants/analysis , Manganese Compounds/chemistry , Colorimetry , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Oxidation-Reduction , Nanostructures/chemistry , Catalysis
5.
Protein Sci ; 33(6): e5014, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38747384

ABSTRACT

A heterodisulfide reductase-like complex (sHdr) and novel lipoate-binding proteins (LbpAs) are central players of a wide-spread pathway of dissimilatory sulfur oxidation. Bioinformatic analysis demonstrate that the cytoplasmic sHdr-LbpA systems are always accompanied by sets of sulfur transferases (DsrE proteins, TusA, and rhodaneses). The exact composition of these sets may vary depending on the organism and sHdr system type. To enable generalizations, we studied model sulfur oxidizers from distant bacterial phyla, that is, Aquificota and Pseudomonadota. DsrE3C of the chemoorganotrophic Alphaproteobacterium Hyphomicrobium denitrificans and DsrE3B from the Gammaproteobacteria Thioalkalivibrio sp. K90mix, an obligate chemolithotroph, and Thiorhodospira sibirica, an obligate photolithotroph, are homotrimers that donate sulfur to TusA. Additionally, the hyphomicrobial rhodanese-like protein Rhd442 exchanges sulfur with both TusA and DsrE3C. The latter is essential for sulfur oxidation in Hm. denitrificans. TusA from Aquifex aeolicus (AqTusA) interacts physiologically with AqDsrE, AqLbpA, and AqsHdr proteins. This is particularly significant as it establishes a direct link between sulfur transferases and the sHdr-LbpA complex that oxidizes sulfane sulfur to sulfite. In vivo, it is unlikely that there is a strict unidirectional transfer between the sulfur-binding enzymes studied. Rather, the sulfur transferases form a network, each with a pool of bound sulfur. Sulfur flux can then be shifted in one direction or the other depending on metabolic requirements. A single pair of sulfur-binding proteins with a preferred transfer direction, such as a DsrE3-type protein towards TusA, may be sufficient to push sulfur into the sink where it is further metabolized or needed.


Subject(s)
Bacterial Proteins , Oxidation-Reduction , Oxidoreductases , Sulfur , Sulfurtransferases , Sulfur/metabolism , Sulfurtransferases/metabolism , Sulfurtransferases/chemistry , Sulfurtransferases/genetics , Oxidoreductases/metabolism , Oxidoreductases/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics
6.
Nat Commun ; 15(1): 4158, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38755143

ABSTRACT

Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function.


Subject(s)
Ascorbic Acid , Evolution, Molecular , Phylogeny , Ascorbic Acid/biosynthesis , Ascorbic Acid/metabolism , Kinetics , Oxidoreductases/metabolism , Oxidoreductases/genetics , Oxidoreductases/chemistry , Crystallography, X-Ray , Oxidation-Reduction , Animals , Catalytic Domain , Substrate Specificity , Models, Molecular
7.
Mikrochim Acta ; 191(6): 352, 2024 05 28.
Article in English | MEDLINE | ID: mdl-38806756

ABSTRACT

Developing convenient and reliable methods for Hg2+ monitoring is highly important. Some precious metal nanomaterials with intriguing peroxidase-like activity have been used for highly sensitive Hg2+ detection. However, H2O2 must be added during these detections, which impedes practical applications of Hg2+ sensors due to its susceptible decomposition by environmental factors. Herein, we discovered that the combination of Hg2+ and palladium metal-organic framework@graphene (Pd-MOF@GNs) exhibits oxidase-like activity (OXD). In the absence of H2O2, this activity not only catalyzes the oxidation of chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) or o-phenylenediamine (OPD) to produce a color change but also enhances the electrical signals during OPD oxidation. Based on these properties, an effective and convenient dual-mode colorimetric and electrochemical sensor for Hg2+ has been developed. The colorimetric and amperometric linear relationships for Hg2+ were 0.045 µM-0.25 mM and 0.020 µM-2.0 mM, respectively. The proposed strategy shows good recovery in real sample tests, indicating promising prospects for multiple environmental sample detection of Hg2+ without relying on H2O2. The colorimetric and electrochemical dual-mode Hg2+ sensor is expected to hold great potentials in applications such as environmental monitoring, rapid field detection, and integration into smartphone detection of Hg2+.


Subject(s)
Colorimetry , Electrochemical Techniques , Graphite , Limit of Detection , Mercury , Metal-Organic Frameworks , Palladium , Graphite/chemistry , Colorimetry/methods , Mercury/analysis , Mercury/chemistry , Metal-Organic Frameworks/chemistry , Palladium/chemistry , Electrochemical Techniques/methods , Benzidines/chemistry , Oxidation-Reduction , Water Pollutants, Chemical/analysis , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/analysis , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Phenylenediamines/chemistry
8.
Org Lett ; 26(21): 4463-4468, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38747552

ABSTRACT

(S)-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-1-(4-methoxybenzyl)-OHIQ) is the key intermediate of the nonopioid antitussive dextromethorphan. In this study, (S)-IR61-V69Y/P123A/W179G/F182I/L212V (M4) was identified with a 766-fold improvement in catalytic efficiency compared with wide-type IR61 through enzyme engineering. M4 could completely convert 200 mM of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline into (S)-1-(4-methoxybenzyl)-OHIQ in 77% isolated yield, with >99% enantiomeric excess and a high space-time yield of 542 g L-1 day-1, demonstrating a great potential for the synthesis of dextromethorphan intermediate in industrial applications.


Subject(s)
Dextromethorphan , Dextromethorphan/chemistry , Dextromethorphan/chemical synthesis , Molecular Structure , Oxidoreductases/metabolism , Oxidoreductases/chemistry , Imines/chemistry , Stereoisomerism , Antitussive Agents/chemistry , Antitussive Agents/chemical synthesis , Protein Engineering
9.
J Biol Inorg Chem ; 29(3): 279-290, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38720157

ABSTRACT

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl-. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding.


Subject(s)
Marinobacter , Oxidoreductases , Marinobacter/enzymology , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Copper/chemistry , Copper/metabolism , Models, Molecular , Crystallography, X-Ray
10.
Nat Commun ; 15(1): 4515, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38802430

ABSTRACT

In extant biology, large and complex enzymes employ low molecular weight cofactors such as dihydronicotinamides as efficient hydride transfer agents and electron carriers for the regulation of critical metabolic processes. In absence of complex contemporary enzymes, these molecular cofactors are generally inefficient to facilitate any reactions on their own. Herein, we report short peptide-based amyloid nanotubes featuring exposed arrays of cationic and hydrophobic residues that can bind small molecular weak hydride transfer agents (NaBH4) to facilitate efficient reduction of ester substrates in water. In addition, the paracrystalline amyloid phases loaded with borohydrides demonstrate recyclability, substrate selectivity and controlled reduction and surpass the capabilities of standard reducing agent such as LiAlH4. The amyloid microphases and their collaboration with small molecular cofactors foreshadow the important roles that short peptide-based assemblies might have played in the emergence of protometabolism and biopolymer evolution in prebiotic earth.


Subject(s)
Amyloid , Peptides , Peptides/chemistry , Peptides/metabolism , Amyloid/chemistry , Amyloid/metabolism , Oxidoreductases/metabolism , Oxidoreductases/chemistry , Nanotubes/chemistry , Oxidation-Reduction
11.
Appl Microbiol Biotechnol ; 108(1): 323, 2024 May 07.
Article in English | MEDLINE | ID: mdl-38713233

ABSTRACT

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.


Subject(s)
Aldehydes , Ergot Alkaloids , Aldehydes/metabolism , Aldehydes/chemistry , Catalytic Domain , Ergot Alkaloids/biosynthesis , Ergot Alkaloids/chemistry , Ergot Alkaloids/metabolism , Molecular Docking Simulation , Mutagenesis, Site-Directed , Oxidation-Reduction , Oxidoreductases/metabolism , Oxidoreductases/genetics , Oxidoreductases/chemistry
12.
Colloids Surf B Biointerfaces ; 239: 113953, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38729021

ABSTRACT

Ascorbic acid (AA) is a powerful antioxidant in food safety and disease treatment. It is of great significance to develop a low-cost, high-stability, and easy-to-operate colorimetric method for quantitative detection of AA in food or human body. Although various nanozymes have been developed for the colorimetric detection of AA, the size regulation of the catalytic center of nanozymes remains a challenge. In this work, we propose a combined strategy of flow chemistry synthesis and pyrolysis to realize the controllable adjustment of the catalytic center size of nanozymes. Zinc-cobalt zeolitic imidazole frameworks (ZnCo-ZIFs) with different sizes are synthesized by flow chemistry. Nitrogen-doped carbon materials with different Co catalytic centers (80 nm-10 nm) are then obtained by pyrolysis of ZnCo-ZIFs precursors. Among them, cobalt quantum dot embedded nitrogen-doped carbon (Co QDs/N-C) exhibits excellent oxidase activity, with Vmax and Km of 4.19 × 10-7 M s-1 and 0.12 mM. Therefore, a simple, low-cost, and stable colorimetric method for the detection of AA is established with a good linear relationship (3-500 µM) and low detection limit (0.40 µM). This work has certain guiding significance for the size regulation of catalytic center of nanozyme, and the detection method has broad application prospects in biochemical sensing field.


Subject(s)
Ascorbic Acid , Carbon , Cobalt , Nitrogen , Quantum Dots , Quantum Dots/chemistry , Ascorbic Acid/analysis , Ascorbic Acid/chemistry , Nitrogen/chemistry , Cobalt/chemistry , Carbon/chemistry , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Colorimetry/methods , Particle Size , Limit of Detection , Humans , Surface Properties , Catalysis
13.
J Phys Chem Lett ; 15(22): 5804-5813, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38781458

ABSTRACT

Nanozymes are unique materials with many valuable properties for applications in biomedicine, biosensing, environmental monitoring, and beyond. In this work, we developed a machine learning (ML) approach to search for new nanozymes and deployed a web platform, DiZyme, featuring a state-of-the-art database of nanozymes containing 1210 experimental samples, catalytic activity prediction, and DiZyme Assistant interface powered by a large language model (LLM). For the first time, we enable the prediction of multiple catalytic activities of nanozymes by training an ensemble learning algorithm achieving R2 = 0.75 for the Michaelis-Menten constant and R2 = 0.77 for the maximum velocity on unseen test data. We envision an accurate prediction of multiple catalytic activities (peroxidase, oxidase, and catalase) promoting novel applications for a wide range of surface-modified inorganic nanozymes. The DiZyme Assistant based on the ChatGPT model provides users with supporting information on experimental samples, such as synthesis procedures, measurement protocols, etc. DiZyme (dizyme.aicidlab.itmo.ru) is now openly available worldwide.


Subject(s)
Machine Learning , Catalysis , Catalase/chemistry , Catalase/metabolism , Nanostructures/chemistry , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Peroxidase/chemistry , Peroxidase/metabolism , Algorithms
14.
Anal Methods ; 16(22): 3577-3586, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38787692

ABSTRACT

Analysis of exosomes provides important information for rapid and non-invasive screening of tumors. However, sensitive and convenient detection of exosomes remains technically challenging to date. Herein, a colorimetric aptasensor based on the light-stimulated oxidase-mimicking activity of FITC was constructed for detecting ovarian cancer (OC) exosomes. The aptasensor contained an EpCAM aptamer to capture OC exosomes. Cholesterol and fluorescein (FITC) were used to modify either end of the DNA (DNA anchor). The DNA anchor could combine with exosomes through a hydrophobic reaction between cholesterol and the lipid membrane. FITC oxidized 3,3',5,5'-tetramethylbenzidine (TMB) under a 365 nm LED light source in a temporally controllable manner under mild conditions, causing the solution to change from colorless to blue, and the corresponding UV-vis absorbance increased. Based on this principle, the exosomes were qualitatively analyzed by observing the color change with the naked eye. In parallel, the exosome concentration was also detected using UV-vis spectrophotometry. The linear range was from 2 × 105 to 100 × 105 particles per mL with a limit of detection of 1.77 × 105 particles per mL. The developed aptasensor also exhibited favorable selectivity and could discriminate the exosomes from OC cells and normal cells. Besides, the receiver operating characteristic (ROC) curve demonstrates that it is possible to distinguish between patients with OC and healthy donors (HDs) using exosomes as the biomarker. Our technology may expand the applications of DNA-based detection method-enabled OC diagnostic tools.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Colorimetry , Exosomes , Exosomes/chemistry , Exosomes/metabolism , Humans , Colorimetry/methods , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Female , Ovarian Neoplasms , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Light , Limit of Detection , Fluorescein/chemistry , Benzidines/chemistry , Cell Line, Tumor
15.
J Agric Food Chem ; 72(22): 12685-12695, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38771136

ABSTRACT

Halogenation plays a unique role in the design of agrochemicals. Enzymatic halogenation reactions have attracted great attention due to their excellent specificity and mild reaction conditions. S-adenosyl-l-methionine (SAM)-dependent halogenases mediate the nucleophilic attack of halide ions (X-) to SAM to produce 5'-XDA. However, only 11 SAM-dependent fluorinases and 3 chlorinases have been reported, highlighting the desire for additional halogenases. SAM-dependent hydroxide adenosyltransferase (HATase) has a similar reaction mechanism as halogenases but uses water as a substrate instead of halide ions. Here, we explored a HATase from the thermophile Thermotoga maritima MSB8 and transformed it into a halogenase. We identified a key dyad W8L/V71T for the halogenation reaction. We also obtained the best performing mutants for each halogenation reaction: M1, M2 and M4 for Cl-, Br- and I-, respectively. The M4 mutant retained the thermostability of HATase in the iodination reaction at 80 °C, which surpasses the natural halogenase SalL. QM/MM revealed that these mutants bind halide ions with more suitable angles for nucleophilic attack of C5' of SAM, thus conferring halogenation capabilities. Our work achieved the halide ion specificity of halogenases and generated thermostable halogenases for the first time, which provides new opportunities to expand the halogenase repertoire from hydroxylase.


Subject(s)
Bacterial Proteins , Thermotoga maritima , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Thermotoga maritima/enzymology , Thermotoga maritima/genetics , Thermotoga maritima/chemistry , Halogenation , Substrate Specificity , S-Adenosylmethionine/metabolism , S-Adenosylmethionine/chemistry , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Oxidoreductases/genetics , Biocatalysis
16.
J Agric Food Chem ; 72(22): 12425-12433, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38781442

ABSTRACT

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 µg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.


Subject(s)
Echinochloa , Herbicides , Oxidoreductases , Plant Proteins , Plant Weeds , Pyridazines , Herbicides/pharmacology , Herbicides/chemistry , Pyridazines/pharmacology , Pyridazines/chemistry , Echinochloa/drug effects , Echinochloa/enzymology , Echinochloa/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/chemistry , Plant Proteins/antagonists & inhibitors , Oxidoreductases/genetics , Oxidoreductases/metabolism , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/chemistry , Plant Weeds/drug effects , Plant Weeds/enzymology , Plant Weeds/genetics , Structure-Activity Relationship , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Plant Roots/chemistry , Plant Roots/drug effects , Molecular Structure
17.
J Agric Food Chem ; 72(22): 12832-12841, 2024 Jun 05.
Article in English | MEDLINE | ID: mdl-38785419

ABSTRACT

Capsaicin (CAP) is a primary indicator for assessing the level of pungency. Herein, iron-based single-atom nanozymes (SAzymes) (Fe/NC) with exceptional oxidase-like activity were used to construct an immunosensor for CAP analysis. Fe/NC could imitate oxidase actions by transforming O2 to •O2- radicals in the absence of hydrogen peroxide (H2O2), which could avoid complex operations and unstable results. By regulating the Fe atom loads, an optimal Fe0.7/NC atom usage rate could improve the catalytic activity (Michaelis-Menten constant (Km) = 0.09 mM). Fe0.7/NC was integrated with goat antimouse IgG by facile mix incubation to develop a competitive enzyme-linked immunosorbent assay (ELISA). Our Fe0.7/NC immunosensing platform is anticipated to outperform the conventional ELISA in terms of stability and shelf life. The proposed immunosensor provided color responses across 0.01-1000 ng/mL CAP concentrations, with a detection limit of 0.046 ng/mL. Fe/NC may have potential as nanozymes for CAP detection in spicy foods, with promising applications in food biosensing.


Subject(s)
Biosensing Techniques , Capsaicin , Capsaicin/analysis , Capsaicin/chemistry , Biosensing Techniques/methods , Biosensing Techniques/instrumentation , Oxidoreductases/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Iron/chemistry , Iron/analysis , Limit of Detection , Hydrogen Peroxide/chemistry , Food Analysis/methods
18.
Mikrochim Acta ; 191(5): 296, 2024 05 03.
Article in English | MEDLINE | ID: mdl-38702534

ABSTRACT

A covalent organic framework-based strategy was designed for label-free colorimetric detection of pesticides. Covalent organic framework-based nanoenzyme with excellent oxidase-like catalytic activity was synthesized. Unlike other artificial enzymes, porphyrin-based covalent organic framework (p-COF) as the oxidase mimic showed highly catalytic chromogenic activity and good affinity toward TMB without the presence of H2O2, which can be used as substitute for peroxidase mimics and H2O2 system in the colorimetric reaction. Based on the fact that the pesticide-aptamer complex can inhibit the oxidase activity of p-COF and reduced the absorbance at 650 nm in UV-Vis spectrum, a label-free and facile colorimetric detection of pesticides was designed and fabricated. Under the optimized conditions, the COF-based colorimetric probe for pesticide detection displayed high sensitivity and selectivity. Taking fipronil for example the limit of detection was 2.7 ng/mL and the linear range was 5 -500,000 ng/mL. The strategy was successfully applied to the detection of pesticides with good recovery , which was in accordance with that of HPLC-MS/MS. The COF-based colorimetric detection was free of complicated modification H2O2, which guaranteed the accuracy and reliability of measurements. The COF-based sensing strategy is a potential candidate for the sensitive detection of pesticides of interests.


Subject(s)
Colorimetry , Limit of Detection , Metal-Organic Frameworks , Pesticides , Porphyrins , Colorimetry/methods , Pesticides/analysis , Metal-Organic Frameworks/chemistry , Porphyrins/chemistry , Hydrogen Peroxide/chemistry , Oxidoreductases/chemistry , Aptamers, Nucleotide/chemistry
19.
Food Chem ; 448: 139170, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38579558

ABSTRACT

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.


Subject(s)
Antioxidants , Citrus , Fruit , Manganese , Metal-Organic Frameworks , Oxidoreductases , Temperature , Citrus/chemistry , Citrus/metabolism , Antioxidants/metabolism , Antioxidants/chemistry , Antioxidants/analysis , Fruit/chemistry , Fruit/metabolism , Manganese/metabolism , Manganese/chemistry , Manganese/analysis , Metal-Organic Frameworks/chemistry , Oxidoreductases/metabolism , Oxidoreductases/chemistry
20.
Methods Enzymol ; 696: 231-247, 2024.
Article in English | MEDLINE | ID: mdl-38658081

ABSTRACT

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.


Subject(s)
Biocatalysis , Fluorine , Halogenation , Protein Engineering , Streptomyces , Fluorine/chemistry , Protein Engineering/methods , Streptomyces/enzymology , Streptomyces/genetics , Oxidoreductases/metabolism , Oxidoreductases/genetics , Oxidoreductases/chemistry , Oxidation-Reduction , Nonheme Iron Proteins/chemistry , Nonheme Iron Proteins/metabolism , Nonheme Iron Proteins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry
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