Structure of the iminium reaction intermediate in an engineered aldolase explains the carboligation activity toward arylated ketones and aldehydes.

Two structures of fructose 6-phosphate aldolase, the wild-type and an engineered variant containing five active-site mutations, have been solved by cryoelectron microscopy (cryo-EM). The engineered variant affords production of aldols from aryl substituted ketones and aldehydes. This structure was solved to a resolution of 3.1 Å and contains the critical iminium reaction intermediate trapped in the active site. This provides new information that rationalizes the acquired substrate scope and aids in formulating hypotheses of the chemical mechanism. A Tyr residue (Y131) is positioned for a role as catalytic acid/base during the aldol reaction and the different structures demonstrate mobility of this amino acid residue. Further engineering of this fructose 6-phosphate aldolase (FSA) variant, guided by this new structure, identified additional FSA variants that display improved carboligation activities with 2-hydroxyacetophenone and phenylacetaldehyde.

Rapid preparation of the Amadori rearrangement product of glutamic acid - xylose through intermittent microwave heating and its browning formation potential in microwave thermal processing.

Directional and rapid formation of the Amadori rearrangement product (ARP) from the glutamic acid and xylose was achieved through intermittent microwave heating. The yield of ARP reached 58.09 % by subjecting the system to intermittent microwave heating at a power density of 10 W/g for 14 min. Dehydration rate and microwave effects were found to be key factors to optimize the conditions for directional and rapid preparation of the ARP. Through a comprehensive analysis of the ARP degradation and further browning under both conductive and microwave thermal processing, it was observed that microwave processing significantly accelerated the browning degree of systems, leading to a tenfold reduction in the heating time required for browning. This research presented a promising avenue for the development of novel and expedited methods for the production of ARP and highlighted the potential of ARP in enhancing color quality in fast-cooking applications utilizing microwave.

From femtoseconds to minutes: time-resolved macromolecular crystallography at XFELs and synchrotrons.

Over the last decade, the development of time-resolved serial crystallography (TR-SX) at X-ray free-electron lasers (XFELs) and synchrotrons has allowed researchers to study phenomena occurring in proteins on the femtosecond-to-minute timescale, taking advantage of many technical and methodological breakthroughs. Protein crystals of various sizes are presented to the X-ray beam in either a static or a moving medium. Photoactive proteins were naturally the initial systems to be studied in TR-SX experiments using pump-probe schemes, where the pump is a pulse of visible light. Other reaction initiations through small-molecule diffusion are gaining momentum. Here, selected examples of XFEL and synchrotron time-resolved crystallography studies will be used to highlight the specificities of the various instruments and methods with respect to time resolution, and are compared with cryo-trapping studies.

Metal-Organic Framework Derived Bi-O-Sn/C Nanostructure: Tailoring the Adsorption Site of Dominant Intermediate for Highly Efficient CO2 Electroreduction to Formate.

Electrochemical CO2 reduction into high-value-added formic acid/formate is an attractive strategy to mitigate global warming and achieve energy sustainability. However, the adsorption energy of most catalysts for the key intermediate *OCHO is usually weak, and how to rationally optimize the adsorption of *OCHO is challenging. Here, an effective Bi-Sn bimetallic electrocatalyst (Bi1 -O-Sn1 @C) where a Bi-O-Sn bridge-type nanostructure is constructed with O as an electron bridge is reported. The electronic structure of Sn is precisely tuned by electron transfer from Bi to Sn through O bridge, resulting in the optimal adsorption energy of intermediate *OCHO on the surface of Sn and the enhanced activity for formate production. Thus, the Bi1 -O-Sn1 @C exhibits an excellent Faradaic efficiency (FE) of 97.7% at -1.1 V (vs RHE) for CO2 reduction to formate (HCOO- ) and a high current density of 310 mA cm-2 at -1.5 V, which is one of the best results catalyzed by Bi- and Sn-based catalysts reported previously. Impressively, the FE exceeds 93% at a wide potential range from -0.9 to -1.4 V. In-situ ATR-FTIR, in-situ Raman, and DFT calculations confirm the unique role of the bridge-type structure of Bi-O-Sn in highly efficient electrocatalytic reduction of CO2 into formate.

Intrinsic Molecular Mechanisms of Transformation between Isomeric Intermediates Formed at Different Stages of Cysteine-Xylose Maillard Reaction Model through Dehydration.

2-Threityl-thiazolidine-4-carboxylic acid (TTCA) and Amadori rearrangement product (ARP), the isomeric intermediates derived from the cysteine-xylose (Cys-Xyl) Maillard reaction model, possessed the ability to produce similar flavor profile during the thermal process, but the flavor formation or browning rate of heated TTCA was significantly lower than that of ARP. Macroscopically, the yield of TTCA reached the maximum when the moisture content of the reaction system just dropped to nearly 0% during the thermal reaction-vacuum dehydration process. During the subsequent dynamic intramolecular dehydration process, the reaction remained at an early stage of the Maillard reaction, and TTCA was the main intermediate. Thereinto, the water activity of the samples decreased with the increased dehydration time. From a molecular perspective, the dissipation of free water promoted the conversion of combined water to immobilized water and free water, increasing the intramolecular dehydration. Instantaneous high-temperature dehydration during the spray drying process revealed a higher efficiency than the thermal reaction-vacuum dehydration process, which facilitated the specific conversion of substrates to intermediates (TTCA, ARP). The loss of free water and immobilized water was a key driving force for the direct formation of TTCA/ARP, regulating the formation stages of MRIs. The increase of the inlet air temperature could alter the ratio of TTCA and ARP at the equilibrium state.

Characterization of aminoacrylate intermediates of pyridoxal-5'-phosphate dependent enzymes.

Pyridoxal-5'-phosphate (PLP) Schiff's bases of 2-aminoacrylate are intermediates in ß-elimination and ß-substitution reaction of PLP-dependent enzymes. These enzymes are found in two major families, the α-, or aminotransferase, superfamily, and the ß-family. While the α-family enzymes primarily catalyze ß-eliminations, the ß-family enzymes catalyze both ß-elimination and ß-substitution reactions. Tyrosine phenol-lyase (TPL), which catalyzes the reversible elimination of phenol from l-tyrosine, is an example of an α-family enzyme. Tryptophan synthase catalyzes the irreversible formation of l-tryptophan from l-serine and indole, and is an example of a ß-family enzyme. The identification and characterization of aminoacrylate intermediates in the reactions of both of these enzymes is discussed. The use of UV-visible absorption and fluorescence spectroscopy, X-ray and neutron crystallography, and NMR spectroscopy to identify aminoacrylate intermediates in these and other PLP enzymes is presented.

[Visualization of Enzymatic Reaction by Time-resolved Structural Analysis with Photosensitive Caged Substrate].

Elucidation of molecular mechanism for metalloeznyme-catalyzed reactions is longstanding topics in the bio-inorganic chemistry field, since metalloeznymes play pivotal roles in various biological processes and catalyze reactions with high efficiency under mild conditions. Structural determination of reaction intermediates is key to visualizing the reactions catalyzed by metalloenzymes. Although it is not easy to determine the structure of the transient species by conventional crystallography, newly developed time-resolved X-ray crystallography using X-ray free electron laser (XFEL) has a great potential for the structural characterization of intermediates. However, XFEL-based time-resolved crystallography requires a photo-trigger, hampering its application to non-photosensitive proteins like metalloenzymes. Here, to overcome this issue, we focused on caged substrates, which produce the substrate upon photo-irradiation, to introduce the photo-trigger into the reaction catalyzed by the metalloenzyme. To demonstrate the usefulness of caged substrates for the trigger of XFEL-based time-resolved crystallography, soluble nitric oxide (NO) reductase, which catalyzes the reduction of NO to nitrous oxide (N2O) at a heme active center, and caged NO were used as a model system. Time-resolved spectroscopic analysis showed that the photolysis of caged NO could initiate NO reduction by P450nor in the micro-crystals. Time-resolved crystallography using XFEL enabled us to determine the structures of two intermediates; NO-bound form and subsequent NO-activated form, which provided a unique opportunity to draw the complete picture of the reaction cycle of P450nor. Thus, the combination of caged substrate and XFEL-based time-resolved crystallography is an invaluable method for the visualization of the reactions catalyzed by metalloenzyems.

Discovery of N-Phenylaminomethylthioacetylpyrimidine-2,4-diones as Protoporphyrinogen IX Oxidase Inhibitors through a Reaction Intermediate Derivation Approach.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is an effective target for green herbicide discovery. In this work, we reported the unexpected discovery of a novel series of N-phenylaminomethylthioacetylpyrimidine-2,4-diones (2-6) as promising PPO inhibitors based on investigating the reaction intermediates of our initially designed N-phenyluracil thiazolidinone (1). An efficient one-pot procedure that gave 41 target compounds in good to high yields was developed. Systematic Nicotiana tabacum PPO (NtPPO) inhibitory and herbicidal activity evaluations led to identifying some compounds with improved NtPPO inhibition potency than saflufenacil and good post-emergence herbicidal activity at 37.5-150 g of ai/ha. Among these analogues, ethyl 2-((((2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl)phenyl)amino)methyl)thio)acetate (2c) (Ki = 11 nM), exhibited excellent weed control at 37.5-150 g of ai/ha and was safe for rice at 150 g of ai/ha, indicating that compound 2c has the potential to be developed as a new herbicide for weed management in paddy fields. Additionally, our molecular simulation and metabolism studies showed that the side chains of compound 2c could form a hydrogen-bond-mediated seven-membered ring system; substituting a methyl group at R1 could reinforce the hydrogen bond of the ring system and reduce the metabolic rate of target compounds in planta.

Kinetics model of DNA double-strand break repair in eukaryotes.

This manuscript outlines the kinetics of two main repair pathways of DNA double-strand break (DSB) in eukaryotes: non-homologous end joining (NHEJ) and homologous recombination repair (HRR). In this review, we discuss the precise study of recruitment kinetics of repair proteins based on the latest technologies in the past two decades. Then we simulate the theoretical description of the DNA repair process by mathematical models. In our study, the consecutive reactions chain (CRC) model and continuous-time random walk (CTRW) model have been unified by us, so that we can obtain the function of the number of intermediates with time in the same framework of equations, overcome the incompatibility between the two models. On this basis, we propose a data fitting workflow using these both models. Finally, we give an overview of different real-time quantitative methods and the new mechanism complexity that can be found from the corresponding dynamic models.

Removal of Emerging Pollutants from Water Using Environmentally Friendly Processes: Photocatalysts Preparation, Characterization, Intermediates Identification and Toxicity Assessment.

Pharmaceuticals and pesticides are emerging contaminants problematic in the aquatic environment because of their adverse effects on aquatic life and humans. In order to remove them from water, photocatalysis is one of the most modern technologies to be used. First, newly synthesized photocatalysts were successfully prepared using a sol-gel method and characterized by different techniques (XRD, FTIR, UV/Vis, BET and SEM/EDX). The photocatalytic properties of TiO2, ZnO and MgO nanoparticles were examined according to their removal from water for two antibiotics (ciprofloxacin and ceftriaxone) and two herbicides (tembotrione and fluroxypyr) exposed to UV/simulated sunlight (SS). TiO2 proved to be the most efficient nanopowder under UV and SS. Addition of (NH4)2S2O8 led to the faster removal of both antibiotics and herbicide fluroxypyr. The main intermediates were separated and identified for the herbicides and antibiotic ciprofloxacin. Finally, the toxicity of each emerging pollutant mixture and formed intermediates was assessed on wheat germination and biomass production.

Surface Lattice Oxygen Activation on Sr2Sb2O7 Enhances the Photocatalytic Mineralization of Toluene: from Reactant Activation, Intermediate Conversion to Product Desorption.

Transition-metal oxide photocatalysis has attracted increasing attention in environmental remediation and solar energy conversion. Surface lattice oxygen is the key active site on the metal oxide, but its role and activation mechanism in the photocatalytic VOC mineralization are still unclear. In this work, we have demonstrated that Sr2Sb2O7 exhibits an excellent photocatalytic activity and stability compared to TiO2 (P25) in gaseous toluene mineralization because the lattice oxygen on Sr2Sb2O7 can be activated efficiently. The lattice oxygen of Sr2Sb2O7 promotes the adsorption and activation of O2 and H2O molecules and enhances the production of •O2- and •OH radicals, as confirmed by the electron spin resonance and DFT calculations. The in situ diffuse reflectance infrared Fourier transform spectroscopy spectra are applied to dynamically monitor the intermediate activation and selective conversion. Combined with DFT calculation, the role and the mechanism of lattice oxygen in photocatalysis have been revealed. Owing to the promoted surface lattice oxygen, the selectivity for benzoic acid formation is enhanced and final product desorption is promoted, which could largely advance the ring opening and mineralization of toluene. This work reveals the origin of lattice oxygen activation and the role for efficient VOC degradation at the atomic scale.

Two-step reaction mechanism reveals new antioxidant capability of cysteine disulfides against hydroxyl radical attack.

Cysteine disulfides, which constitute an important component in biological redox buffer systems, are highly reactive toward the hydroxyl radical (•OH). The mechanistic details of this reaction, however, remain unclear, largely due to the difficulty in characterizing unstable reaction products. Herein, we have developed a combined approach involving mass spectrometry (MS) and theoretical calculations to investigate reactions of •OH with cysteine disulfides (Cys-S-S-R) in the gas phase. Four types of first-generation products were identified: protonated ions of the cysteine thiyl radical (+Cys-S•), cysteine (+Cys-SH), cysteine sulfinyl radical (+Cys-SO•), and cysteine sulfenic acid (+Cys-SOH). The relative reaction rates and product branching ratios responded sensitively to the electronic property of the R group, providing key evidence to deriving a two-step reaction mechanism. The first step involved •OH conducting a back-side attack on one of the sulfur atoms, forming sulfenic acid (-SOH) and thiyl radical (-S•) product pairs. A subsequent H transfer step within the product complex was favored for protonated systems, generating sulfinyl radical (-SO•) and thiol (-SH) products. Because sulfenic acid is a potent scavenger of peroxyl radicals, our results implied that cysteine disulfide can form two lines of defense against reactive oxygen species, one using the cysteine disulfide itself and the other using the sulfenic acid product of the conversion of cysteine disulfide. This aspect suggested that, in a nonpolar environment, cysteine disulfides might play a more active role in the antioxidant network than previously appreciated.

Viscous Media Slow Down the Decay of the Key Intermediate in Bacterial Bioluminescent Reaction.

The effects of medium viscosity on the decay rate of the 4a-hydroperoxyflavin intermediate of the bioluminescent reaction was investigated. It was found that at low concentrations of glycerol or sucrose (viscosity 1.1-1.3 cP) the decay rate rises, whereas a further increase in viscosity to 6.2 cP leads to a decrease in the decay rate following a power function with an exponent of 0.82-0.84. Using molecular dynamics methods, it was shown that the presence of glycerol and sucrose molecules causes a change in the mobility of the amino acid residues in the active center of luciferase, particularly those responsible for binding of flavin. The results obtained are indicative of two opposite effects of viscous media with glycerol and sucrose: (1) destabilization of 4a-hydroperoxyflavin due to a change in the structural and dynamic properties of the protein and (2) stabilization of this intermediate by the decrease in the diffusion rate of its decay products.

Aqueous Preparation of Maillard Reaction Intermediate from Glutathione and Xylose and its Volatile Formation During Thermal Treatment.

Maillard reaction intermediate (MGX) generated from glutathione and xylose in aqueous medium was prepared via the Maillard reaction performed under a two-stage temperature increase process. The purified MGX was identified by Fourier-transform infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance as N-(1-deoxy-d-xylulos-1-yl)-glutathione (Amadori compound, C15 H25 O10 N3 S) with five main isomers. The method of Maillard reaction performed under a two-stage temperature increase process was further verified by high-performance liquid chromatography. The optimal reaction time and temperature for the preparation of MGX was determined as 60 min at 90 °C. The yield of MGX was increased from 8.60% to 55.52% through thermal reaction coupled with vacuum dehydration. In addition, rapid and more Maillard-type volatile compounds were formed in MGX during thermal treatment than that in Maillard reaction products or glutathione-xylose mixture. Beside, MGX possessed more pleasing meat-like volatile profile compared with the Amadori compound of glutamic acid-xylose (AAX), cysteine-xylose (ACX), and glycine-xylose (AGX). Therefore, it suggested that the MGX had the potential to achieve a better flavor formation during thermal treatment. PRACTICAL APPLICATION: Maillard reaction intermediates, such as Amadori or Heyns rearrangement products (ARP or HRP), are important flavor precursors, which possess stable physicochemical properties, but tend to degrade into flavor compounds at high temperatures. Maillard reaction intermediate from glutathione and xylose acts as primary flavor enhancers to complete Maillard reaction to produce flavors in the subsequent thermal processing, which can significantly improve and stabilize the flavor quality of the meaty food, and deserves a very broad application prospects. The new developed method will be a significant theoretical basis on research preparation and properties of Maillard reaction intermediates in complex food systems.

Interaction of Added l-Cysteine with 2-Threityl-thiazolidine-4-carboxylic Acid Derived from the Xylose-Cysteine System Affecting Its Maillard Browning.

The Maillard reaction under a stepwise increase of temperature using l-cysteine as the indicator was performed to determine the formation conditions for the preparation of 2-threityl-thiazolidine-4-carboxylic acid (TTCA) which was the main Maillard reaction intermediate (MRI) derived from the xylose (Xyl)-cysteine (Cys) model system in aqueous medium. To clarify the indicating mechanism of Cys for the TTCA formation, the thermal model systems of TTCA-Cys and TTCA solutions were investigated. The browning of the final Maillard reaction products (MRPs) and concentration of downstream degradation products of MRIs indicated that the added Cys could react with TTCA to inhibit the formation of visible color via preventing the generation of dicarbonyl compounds derived from MRIs. Through HPLC analysis, it was demonstrated that added Cys affected the normal reaction pathway from TTCA to ARP and other downstream products by restoring TTCA to sugar and amino acid under heat treatment.

Antioxidant Activity In Vitro of N-(1-deoxy-α-d-xylulos-1-yl)-Phenylalanine: Comparison Among Maillard Reaction Intermediate, End-Products and Xylose-Phenylalanine.

The Maillard reaction end-products (MRPs) and intermediate (MRI, N-(1-deoxy-α-D-xylulos-1-yl)-phenylalanine) derived from xylose (Xyl) and phenylalanine (Phe) model system were synthesized in an aqueous medium, and their antioxidant activity was evaluated. Both the MRPs and N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine exhibited the Fe2+ chelating activity at their addition concentration of 2 to 12 mg/mL. While their reducing power, free radical scavenging activity, and inhibition effect on lipid peroxidation were observed at a lower addition concentration of only 0.2 to 1.2 mg/mL. The free radical scavenging activity was improved with the increase in their concentration, and a time-dependent scavenging effect of the MRI was also shown in the results. However, a weak chelating activity of Xyl-Phe on Fe2+ in a concentration-dependent manner was observed, but no significant free radical scavenging activity or reducing power of Xyl-Phe was found. Based on these results, possible free radical scavenging pathway of the MRI was proposed. As the MRI showed lower antioxidant activity than MRPs, the application of the MRI in combination with MRPs as an antioxidant was proposed in cooked or semi-processed foods to avoid the deterioration induced by oxidation. PRACTICAL APPLICATION: The Maillard reaction intermediates (MRIs) showed antioxidant activity and could be applied in foods as both antioxidants and flavor enhancer. The MRIs will probably extend the shelf-life of lipid rich foods and could be the substitute of artificial antioxidants.

Differentiation between Enamines and Tautomerizable Imines Oxidation Reaction Mechanism using Electron-Vibration-Vibration Two Dimensional Infrared Spectroscopy.

Intermediates lie at the center of chemical reaction mechanisms. However, detecting intermediates in an organic reaction and understanding its role in reaction mechanisms remains a big challenge. In this paper, we used the theoretical calculations to explore the potential of the electron-vibration-vibration two-dimensional infrared (EVV-2DIR) spectroscopy in detecting the intermediates in the oxidation reactions of enamines and tautomerizable imines with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). We show that while it is difficult to identify the intermediates from their infrared and Raman signals, the simulated EVV-2DIR spectra of these intermediates have well resolved spectral features, which are absent in the signals of reactants and products. These characteristic spectral signatures can, therefore, be used to reveal the reaction mechanism as well as monitor the reaction progress. Our work suggests the potential strength of EVV-2DIR technique in studying the molecular mechanism of organic reactions in general.

Expression and Characterization of the Human Intestinal Bacterial Enzyme Which Cleaves the C-Glycosidic Bond in 3″-Oxo-puerarin.

Puerarin (daidzein 8-C-glucoside) is an isoflavone C-glucoside contained in the roots of Pueraria lobata OHWI. We have previously isolated the human intestinal bacterium, strain PUE, which metabolizes puerarin to daidzein, though the enzyme which cleaves C-glycosidic bond has not been clarified. Here, we identified one of the intermediates of enzymatic puerarin C-deglycosylation reaction as 3″-oxo-puerarin (1): C-3 in the glucose moiety connecting to hydroxyl is oxidized to ketone group. 1 was easily isomerized to the mixture of 1, 2″-oxo-puerarin (2a) and cyclic acetal (2b) of 2a in non-enzymatic condition. We identified the putative puerarin-metabolizing operon of strain PUE composed of 8 genes (dgpA-H). Among them, DgpB-C complex was expressed in Escherichia coli, which cleaved the C-glycosidic bond in 1 but not puerarin. These results suggested that the puerarin C-deglycosylation reaction is a two-step enzymatic reaction, including the oxidation reaction at C-3″ in puerarin to give 1, and the subsequent C-deglycosylation of 1 to provide daidzein.

Improved controlled flavor formation during heat-treatment with a stable Maillard reaction intermediate derived from xylose-phenylalanine.

The Maillard reaction intermediate (MRI) and Maillard reaction products (MRPs) derived from xylose (Xyl) and phenylalanine (Phe) were prepared, then stored at 25 °C for 60 days. After storage, the contents of flavor compounds and the clarity of MRPs solution decreased, and the apparent Z-average hydrodynamic diameter (Dh) of particles in the solution increased from 149 to 439 nm. However, the MRI solution remained transparent during storage. The concentration of MRI only decreased by 6.49%, and A294 of the solution increased slightly yet A420 remained stable. Numerous flavor compounds in MRPs decreased during heat treatment, meanwhile the cross-linking and aggregation of MRPs were intensified, and the particles' Dh increased to micron level. The heated MRI solution showed a similar appealing profile and flavor fingerprints as the MRPs solution before heat treatment. Controlled formation of flavors from MRIs is proposed to be used as potential alternative to the existing Maillard flavorings.

X-Ray Crystallography of Carbon Monoxide Dehydrogenases.

Carbon monoxide dehydrogenases (CODHs) are central players in the biogeochemical carbon monoxide (CO) cycle and have been extensively studied from the ecological level to the structural/molecular level. Of the two types of CODHs, the oxygen-tolerant CODHs use a bimetallic [CuSMo(=O)OH] center connected to the protein via a pyranopterin cofactor, whereas the oxygen-sensitive CODHs contain a [NiFe4S4-OHx]-cluster. Despite the fact that we have a basic understanding of how both types of CODHs use distinct active sites to catalyze the oxidation of CO with water to CO2, two protons, and two electrons (a reversible reaction in the cases of the oxygen-sensitive CODHs), many questions remain unanswered, especially concerning the electronic structures of the intermediate states. Since these states will likely be only revealed by the interplay of experimental and theoretical methods, there is a need to obtain accurate descriptions of the active site architectures in various states and, consequently, a need to generate crystals with good diffraction quality and collect data at element-specific wavelengths in order to determine the identity of elements in the case of mixed states. This chapter provides a description of the general working protocols for the crystallization and structural analysis of Cu,Mo-CODH and Ni,Fe-CODH that facilitates the mechanistic investigations of these important metalloenzymes.