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We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The aim of the presented study was to evaluate the release of the enzymatically initiated production of hexanal from double emulsion electrospun bio-active membranes at a temperature of fruit storage. Among different formulations of water-in-oil (W1/O) primary emulsions, the emulsion composed of 12% w/v Tween20 and 0.1 M NaCl in water (W1) and 6% of poly(glycerol) poly(ricinoleate) dissolved in sunflower oil (O) using W1/O ratio of 80/20 (w/w) (Tween20-NaCl/6% PGPR) was selected, for further incorporation of enzymes, based on the lowest average droplet size (391.0 ± 15.6 nm), low polydispersity index (0.255 ± 0.07), and good gravitational stability also after 14 days. Both enzymes, lipase and lipoxygenase are needed to produce hexanal (up to 58 mg/L). Additionally, double emulsions were prepared with sufficient conductivity and viscosity using different W1/O to W2 ratios for electrospinning. From the selected electrospun membrane, up to 4.5 mg/L of hexanal was released even after 92 days., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lipoxygenases (LOXs) from lower organisms have substrate flexibility and function versatility in fatty acid oxidation, but it is not clear how these LOXs acquired the ability to execute multiple functions within only one catalytic domain. This work studied a multifunctional LOX from red alga Pyropia haitanensis (PhLOX) which combined hydroperoxidelyase (HPL) and allene oxide synthase (AOS) activity in its active pocket. Molecular docking and site-directed mutagenesis revealed that Phe642 and Phe826 jointly regulated the double peroxidation of fatty acid, Gln777 and Asn575 were essential to the AOS function, and the HPL activity was improved when Asn575, Gln777, or Phe826 was replaced by leucine. Phylogenetic analysis indicated that Asn575 and Phe826 were unique amino acid sites in the separated clades clustered with PhLOX, whereas Phe642 and Gln777 were conserved in plant or animal LOXs. The N-terminal START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) domain of PhLOX was another key variable, as the absence of this domain disrupted the versatility of PhLOX. Moreover, the functions of two homologous LOXs from marine bacterium Shewanella violacea and red alga Chondrus crispus were examined. The HPL activity of PhLOX appeared to be inherited from a common ancestor, and the AOS function was likely acquired through mutations in some key residues in the active pocket. Taken together, our results suggested that some LOXs from red algae attained their versatility by amalgamating functional domains of ancestral origin and unique amino acid mutations.

The structural and digestive properties of indica rice starch-fatty acid complexes and the effects of lipoxygenase on the structural and digestive properties of the complexes were examined in this study. The complexes were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform-infrared spectroscopy and Raman spectroscopy. The results showed that indica rice starch had the highest molecular chain order and the highest crystallinity, and the crystallization disappeared after gelatinization, and the formation of indica rice starch-fatty acid complexes promoted the transformation of starch crystal structure from A-type to V-type. Lipoxygenase reduced the regularity of starch molecular crystal structure in the complexes, while enzyme protein improved the order of starch molecular structure in the complexes. The regularity of starch crystal structure in the complexes could improve with the increase of composite temperature and the increase of fatty acid unsaturation. In vitro digestibility and in vitro digestion kinetics showed that the formation of indica rice starch-fatty acid complexes reduced the digestibility of indica rice starch to a certain extent. The RDS content of indica rice starch was 66.42 ± 0.39 %, and lipoxygenase reduced the reduction of rapidly digested starch content during complexes digestion, while enzyme protein increased the content of resistant starch., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Manganese hydroxido (Mn-OH) complexes supported by a tripodal N,N',N″-[nitrilotris(ethane-2,1-diyl)]tris(P,P-diphenylphosphinic amido) ([poat] 3- ) ligand have been synthesized and characterized by spectroscopic techniques including UV-vis and electron paramagnetic resonance (EPR) spectroscopies. X-ray diffraction (XRD) methods were used to confirm the solid-state molecular structures of {Na 2 [Mn II poat(OH)]} 2 and {Na[Mn III poat(OH)]} 2 as clusters that are linked by the electrostatic interactions between the sodium counterions and the oxygen atom of the ligated hydroxido unit and the phosphinic (P=O) amide groups of [poat] 3- . Both clusters feature two independent monoanionic fragments in which each contains a trigonal bipyramidal Mn center that is comprised of three equatorial deprotonated amide nitrogen atoms, an apical tertiary amine, and an axial hydroxido ligand. XRD analyses of {Na[Mn III poat(OH)]} 2 also showed an intramolecular hydrogen bonding interaction between the Mn III -OH unit and P=O group of [poat] 3- . Crystalline {Na[Mn III poat(OH)]} 2 remains as clusters with Na + ---O interactions in solution and is unreactive toward external substrates. However, conductivity studies indicated that [Mn III poat(OH)] - generated in situ is monomeric and reactivity studies found that it is capable of cleaving C-H bonds, illustrating the importance of solution-phase speciation and its direct effect on chemical reactivity. Synopsis: Manganese-hydroxido complexes were synthesized to study the influence of H-bonds in the secondary coordination sphere and their effects on the oxidative cleavage of substrates containing C-H bonds., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Inflammation is the preliminary response given to any possible harmful stimuli including infections, injury or stress by immune system where neutrophils and macrophages gets activated and produces mediators, such as nitric oxide and cytokines that serves as biomarkers of inflammation. Lipoxygenases are enzymes that peroxidises lipids and are involved in the pathogenesis of several diseases including inflammatory diseases. These are oxidative enzymes comprising a non-heme iron atom in active site and are convoluted in inflammatory reactions. Fucoidan is sulphated polysaccharide that has numerous pharmacological implications. Implications of fucoidan on inflammatory diseases are still an objective of rigorous research. Therefore, this study focusses on investigating lipoxygenase inhibitory activities of fucoidan. The mechanism of lipoxygenase inhibitory activities of fucoidan was studied via molecular docking and molecular dynamics simulations. The docking score produced by the binding of the fucoidan to the lipoxygenase was - 6.69 kcal/mol whereas, the docking score in case of Aspirin and Zileuton were -5.8 kcal/mol and -7.0 kcal/mol and it was found that fucoidan makes hydrogen bonds with lipoxygenase protein through polar amino acid glutamine at GLN 514. The results obtained from molecular dynamics simulations proposed the development of a stable complex between fucoidan and lipoxygenase due to the establishment of favourable interactions with amino acid residues and indicated efficient results when compared with Aspirin and Zileuton. This study suggested that fucoidan had anti-inflammatory potentials and thus can be used as a promising drug candidate against inflammation.Communicated by Ramaswamy H. Sarma.

Lipoxygenases (LOXs) from pathogenic fungi are potential therapeutic targets for defense against plant and select human diseases. In contrast to the canonical LOXs in plants and animals, fungal LOXs are unique in having appended N -linked glycans. Such important post-translational modifications (PTMs) endow proteins with altered structure, stability, and/or function. In this study, we present the structural and functional outcomes of removing or altering these surface carbohydrates on the LOX from the devastating rice blast fungus, M. oryzae , Mo LOX. Alteration of the PTMs did notinfluence the active site enzyme-substrate ground state structures as visualized by electron-nuclear double resonance (ENDOR) spectroscopy. However, removal of the eight N -linked glycans by asparagine-to-glutamine mutagenesis nonetheless led to a change in substrate selectivity and an elevated activation energy for the reaction with substrate linoleic acid, as determined by kinetic measurements. Comparative hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of wild-type and Asn-to-Gln Mo LOX variants revealed a regionally defined impact on the dynamics of the arched helix that covers the active site. Guided by these HDX results, a single glycan sequon knockout was generated at position 72, and its comparative substrate selectivity from kinetics nearly matched that of the Asn-to-Gln variant. The cumulative data from model glyco-enzyme Mo LOX showcase how the presence, alteration, or removal of even a single N -linked glycan can influence the structural integrity and dynamics of the protein that are linked to an enzyme's catalytic proficiency, while indicating that extensive glycosylation protects the enzyme during pathogenesis by protecting it from protease degradation.

This Chapter describes methods for the biosynthetic substitution of the mononuclear, non-heme iron in plant and animal lipoxygenases (LOXs). Substitution of this iron center for a manganese ion results in an inactive, yet faithful structural surrogate of the LOX enzymes. This metal ion substitution permits structural and dynamical studies of enzyme-substrate complexes in solution and immobilized on lipid membrane surfaces. Representative procedures for two LOXs, soybean lipoxygenase (SLO) from plants and human epithelial 15-lipoxygenase-2 (15-LOX-2) from mammals, are described as examples., (Copyright © 2024. Published by Elsevier Inc.)

Lipoxygenase (LOX) enzymes produce important cell-signaling mediators, yet attempts to capture and characterize LOX-substrate complexes by X-ray co-crystallography are commonly unsuccessful, requiring development of alternative structural methods. We previously reported the structure of the complex of soybean lipoxygenase, SLO, with substrate linoleic acid (LA), as visualized through the integration of 13 C/ 1 H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) computations. However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn 2+ ion as a spin probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear Mn 2+ metallocenters. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from rice blast pathogen Magnaporthe oryzae , Mo LOX complexed with LA, as obtained through the 13 C/ 1 H ENDOR-guided MD approach. The catalytically important distance between the hydrogen donor, carbon-11 (C11), and the acceptor, Mn-bound oxygen, (donor-acceptor distance, DAD) for the Mo LOX-LA complex derived in this fashion is 3.4 ± 0.1 Å. The difference of the Mo LOX-LA DAD from that of the SLO-LA complex, 3.1 ± 0.1 Å, is functionally important, although is only 0.3 Å, despite the Mo LOX complex having a Mn-C11 distance of 5.4 Å and a "carboxylate-out" substrate-binding orientation, whereas the SLO complex has a 4.9 Å Mn-C11 distance and a "carboxylate-in" substrate orientation. The results provide structural insights into reactivity differences across the LOX family, give a foundation for guiding development of Mo LOX inhibitors, and highlight the robustness of the ENDOR-guided MD approach to describe LOX-substrate structures.

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser 600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2 WT ). AtLOX2 WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser 600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2 S600D and Atlox2 S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2 S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser 600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser 600 is located at a critical position within the AtLOX2 structure and highlights how Ser 600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser 600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Formulations of hydrogen tunneling in enzyme-catalysed C-H activation reactions indicate enthalpic barriers to reaction that are independent of chemical steps and dependent on the protein scaffold. A tool to identify catalytically relevant site-specific protein thermal networks has emerged from temperature-dependent hydrogen deuterium exchange (TDHDX). Focusing on mutant enzyme forms with altered activation energies for catalysis, TDHDX provides a comparative analysis of the impact of mutation on E a for local protein unfolding. Identified thermal networks appear unrelated to protein scaffold conservation and track to the dictates of the catalysed reaction, including sites for metal binding. The positions of thermal networks provide a framework for further understanding of time-dependent, functionally relevant protein motions. Measurement of nanosecond Stokes shifts at the surface of the thermal network in soybean lipoxygenase yields activation energies that are identical to E a values measured for k cat . This finding identifies a rapid (> nanosecond), long-range and cooperative structural reorganization as the thermal barrier to catalysis. A model for protein dynamics is put forward that integrates broadly distributed protein conformational sampling with protein embedded thermal networks., (© 2022 Federation of European Biochemical Societies.)

Lipid oxidation will lead to the deterioration of flavor, color and texture of aquatic products with high fatty acid content. The mechanism of ultrasound (US) combined with rosmarinic acid (RA) on lipid oxidation and endogenous enzyme activities of large yellow croaker during cold-storage (4 ℃) was investigated. The result showed that the US and RA have synergistic effects in delaying lipid oxidation and inhibiting endogenous lipase and lipoxygenase (LOX) activities related to oxidation. The inhibition of LOX activity by RA was dose-dependent, and US showed a negative effect on the inhibition of enzyme activity in the presence of low concentration RA. Moreover, RA changes the enzyme structure through static fluorescence quenching and interaction with enzyme molecules. Hydrogen bonding and hydrophobic interaction are the main interaction forces between RA and LOX. This study could provide basic mechanism of US treatment cooperating with polyphenols to inhibit lipid oxidation during food preservation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

5-lipoxygenase (5-LOX) was a key enzyme involved in many inflammatory diseases. Sec-O-glucosylhamaudol (SOG) was a chromone found in Saposhnikovia divaricata (Turcz.) Schischk (S. divaricate). The potato-derived 5-LOX (p-5-LOX) and human recombinant 5-LOX (h-5-LOX) were selected as model protein due to their simple usability and high stability in this study. Thus, the binding interactions of p-5-LOX and h-5-LOX with SOG were investigated by multi-spectroscopy and molecular docking. As a result, the fluorescence intensities of the two 5-LOX were quenched statically by SOG. However, the binding ability of SOG to h-5-LOX was higher than that of p-5-LOX at the same temperature. The results of multi-spectroscopy revealed that the conformation and micro-environment of the two 5-LOX proteins were changed after binding with SOG. Fluorescence assay and molecular docking indicated that hydrogen bond and electrostatic gravitation were the main forces between the two 5-LOX and SOG. Our results here suggested that SOG may exert anti-inflammatory effect by inhibiting 5-LOX activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Previous comparative kinetic isotope effects have inferred an allosteric site for fatty acids and their derivatives that modulates substrate selectivity in 15-lipoxygenases. Hydrogen-deuterium exchange also previously revealed regionally defined enhanced protein flexibility, centred at helix α2 - a gate to the substrate entrance. Direct evidence for allosteric binding and a complete understanding of its mechanism remains elusive. In this study, we examine the binding thermodynamics of the fatty acid mimic, oleyl sulfate (OS), with the monomeric model plant 15-LOX, soybean lipoxygenase (SLO), using isothermal titration calorimetry. Dynamic light scattering and differential scanning calorimetry rule out OS-induced oligomerization or structural changes. These data provide evidence that the fatty acid allosteric regulation of SLO is controlled by the dynamics of helix α2., (© 2022 Federation of European Biochemical Societies.)

Manganese lipoxygenase (MnLOX) is an enzyme that converts polyunsaturated fatty acids to alkyl hydroperoxides. In proposed mechanisms for this enzyme, the transfer of a hydrogen atom from a substrate C-H bond to an active-site Mn III -hydroxo center initiates substrate oxidation. In some proposed mechanisms, the active-site Mn III -hydroxo complex is regenerated by the reaction of a Mn III -alkylperoxo intermediate with water by a ligand substitution reaction. In a recent study, we described a pair of Mn III -hydroxo and Mn III -alkylperoxo complexes supported by the same amide-containing pentadentate ligand ( 6Me dpaq). In this present work, we describe the reaction of the Mn III -hydroxo unit in C-H and O-H bond oxidation processes, thus mimicking one of the elementary reactions of the MnLOX enzyme. An analysis of kinetic data shows that the Mn III -hydroxo complex [Mn III (OH)( 6Me dpaq)] + oxidizes TEMPOH (2,2'-6,6'-tetramethylpiperidine-1-ol) faster than the majority of previously reported Mn III -hydroxo complexes. Using a combination of cyclic voltammetry and electronic structure computations, we demonstrate that the weak Mn III -N(pyridine) bonds lead to a higher Mn III/II reduction potential, increasing the driving force for substrate oxidation reactions and accounting for the faster reaction rate. In addition, we demonstrate that the Mn III -alkylperoxo complex [Mn III (OO t Bu)( 6Me dpaq)] + reacts with water to obtain the corresponding Mn III -hydroxo species, thus mimicking the ligand substitution step proposed for MnLOX.

The enzymes manganese lipoxygenase (MnLOX) and manganese superoxide dismutase (MnSOD) utilize mononuclear Mn centers to effect their catalytic reactions. In the oxidized Mn III state, the active site of each enzyme contains a hydroxo ligand, and X-ray crystal structures imply a hydrogen bond between this hydroxo ligand and a cis carboxylate ligand. While hydrogen bonding is a common feature of enzyme active sites, the importance of this particular hydroxo-carboxylate interaction is relatively unexplored. In this present study, we examined a pair of Mn III -hydroxo complexes that differ by a single functional group. One of these complexes, [Mn III (OH)(PaPy 2 N)] + , contains a naphthyridinyl moiety capable of forming an intramolecular hydrogen bond with the hydroxo ligand. The second complex, [Mn III (OH)(PaPy 2 Q)] + , contains a quinolinyl moiety that does not permit any intramolecular hydrogen bonding. Spectroscopic characterization of these complexes supports a common structure, but with perturbations to [Mn III (OH)(PaPy 2 N)] + , consistent with a hydrogen bond. Kinetic studies using a variety of substrates with activated O-H bonds, revealed that [Mn III (OH)(PaPy 2 N)] + is far more reactive than [Mn III (OH)(PaPy 2 Q)] + , with rate enhancements of 15-100-fold. A detailed analysis of the thermodynamic contributions to these reactions using DFT computations reveals that the former complex is significantly more basic. This increased basicity counteracts the more negative reduction potential of this complex, leading to a stronger O-H BDFE in the [Mn II (OH 2 )(PaPy 2 N)] + product. Thus, the differences in reactivity between [Mn III (OH)(PaPy 2 Q)] + and [Mn III (OH)(PaPy 2 N)] + can be understood on the basis of thermodynamic considerations, which are strongly influenced by the ability of the latter complex to form an intramolecular hydrogen bond.

Verticillium wilt is a major limiting factor for sustainable production of cotton but the mechanism of controlling this disease is still poorly understood. Lipoxygenase (LOX)-derived oxylipins have been implicated in defense responses against diverse pathogens; however there is limited information about the functional characterization of LOXs in response to Verticillium dahliae infection. In this study, we report the characterization of a cotton LOX gene, GhLOX2, which phylogenetically clustered into 13-LOX subfamily and is closely related to Arabidopsis LOX2 gene. GhLOX2 was predominantly expressed in leaves and strongly induced following V. dahliae inoculation and treatment of methyl jasmonate (MeJA). RNAi-mediated knock-down of GhLOX2 enhanced cotton susceptibility to V. dahliae and was coupled with suppression of jasmonic acid (JA)-related genes both after inoculation with the cotton defoliating strain V991 or MeJA treatment. Interestingly, lignin contents, transcripts of lignin synthesis genes and H 2 O 2 contents were also decreased in GhLOX2-silenced plants. This study suggests that GhLOX2 is involved in defense responses against infection of V. dahliae in cotton and supports that JA is one of the major defense hormones against this pathogen., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Iron complexes that model the structural and functional properties of the active iron site in rabbit lipoxygenase are described. The ligand sphere of the mononuclear pseudo-octahedral cis -(carboxylato)(hydroxo)iron(III) complex, which is completed by a tetraazamacrocyclic ligand, reproduces the first coordination shell of the active site in the enzyme. In addition, two corresponding iron(II) complexes are presented that differ in the coordination of a water molecule. In their structural and electronic properties, both the (hydroxo)iron(III) and the (aqua)iron(II) complex reflect well the only two essential states found in the enzymatic mechanism of peroxidation of polyunsaturated fatty acids. Furthermore, the ferric complex is shown to undergo hydrogen atom abstraction reactions with O-H and C-H bonds of suitable substrates, and the bond dissociation free energy of the coordinated water ligand of the ferrous complex is determined to be 72.4 kcal·mol -1 . Theoretical investigations of the reactivity support a concerted proton-coupled electron transfer mechanism in close analogy to the initial step in the enzymatic mechanism. The propensity of the (hydroxo)iron(III) complex to undergo H atom abstraction reactions is the basis for its catalytic function in the aerobic peroxidation of 2,4,6-tri( tert -butyl)phenol and its role as a radical initiator in the reaction of dihydroanthracene with oxygen.

Oxidative stress and inflammation are two conditions that coexist in many multifactorial diseases such as atherosclerosis and neurodegeneration. Thus, the design of multifunctional compounds that can concurrently tackle two or more therapeutic targets is an appealing approach. In this study, the basic NSAID structure was fused with the antioxidant moieties 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHB), its reduced alcohol 3,5-di-tert-butyl- 4-hydroxybenzyl alcohol (BHBA), or 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), a hydrophilic analogue of α-tocopherol. Machine learning algorithms were utilized to validate the potential dual effect (anti-inflammatory and antioxidant) of the designed analogues. Derivatives 1 - 17 were synthesized by known esterification methods, with good to excellent yields, and were pharmacologically evaluated both in vitro and in vivo for their antioxidant and anti-inflammatory activity, whereas selected compounds were also tested in an in vivo hyperlipidemia protocol. Furthermore, the activity/binding affinity of the new compounds for lipoxygenase-3 (LOX-3) was studied not only in vitro but also via molecular docking simulations. Experimental results demonstrated that the antioxidant and anti-inflammatory activities of the new fused molecules were increased compared to the parent molecules, while molecular docking simulations validated the improved activity and revealed the binding mode of the most potent inhibitors. The purpose of their design was justified by providing a potentially safer and more efficient therapeutic approach for multifactorial diseases.

Natural product is a well-known source of bioactive compounds. Herein, a steroidal compound stigmasta-7,22-diene-3-one (stigmastadienone) has been isolated from Isodon rugosus. The potency of isolated compound has been tested for several in-vitro targets. The acetyl and butyrylcholinesterase assays were performed using Ellman's procedure. For the in-vitro antidiabetic potential, α-glucosidase inhibitory assay was performed. Similarly, the cyclo and lipoxygenase pathways were studied to find its potential role in the management of inflammation and analgesia. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide (H 2 O 2 ) assays were performed for the antioxidant potentials. Docking studies were performed against acetylcholinesterase, cyclooxygenase and lipoxygenase targets. In anticholinesterase assays, stigmastadienone exhibited half-maximal inhibitory concentration (IC 50 ) values of 13.52 and 11.53 μg/ml for acetyl and butyrylcholinesterase respectively. The observed IC 50 values for that of galantamine were 6.07 and 4.42 μg/ml for acety and butyrylcholinesterase respectively. In inhibiting α-glucosidase enzyme, the compound showed mediocre IC 50 of 109.40 μg/ml compared to the standard acarbose (7.60 μg/ml). The stigmastadienone proved to be an excellent inhibitor of cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX) attaining IC 50 values of 4.72 and 3.36 μg/ml respectively. The standard drugs IC 50 values for COX-2 (celecoxib) and 5-LOX (montelukast) were 3.81 and 2.74 μg/ml respectively. The enzymatic activities of stigmastadienone were also supplemented with antioxidant results, specifically it was more dominant against DPPH and ABTS free radicals. Docking studies showed that only the carbonyl oxygen is able to form hydrogen bond interaction with the residues. In conclusions, the stigmastadienone has been isolated from Isodon rugosus for the first time. Moreover, the compound has been evaluated for several biochemical pathways which suggest its pharmacological role on the explored targets., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Radiofrequency ablation (RFA) is clinically adopted to destruct solid tumors, but is often incapable of completely ablating large tumors and those with multiple metastatic sites. Here we develop a CaCO 3 -assisted double emulsion method to encapsulate lipoxidase and hemin with poly(lactic-co-glycolic acid) (PLGA) to enhance RFA. We show the HLCaP nanoreactors (NRs) with pH-dependent catalytic capacity can continuously produce cytotoxic lipid radicals via the lipid peroxidation chain reaction using cancer cell debris as the fuel. Upon being fixed inside the residual tumors post RFA, HLCaP NRs exhibit a suppression effect on residual tumors in mice and rabbits by triggering ferroptosis. Moreover, treatment with HLCaP NRs post RFA can prime antitumor immunity to effectively suppress the growth of both residual and metastatic tumors, also in combination with immune checkpoint blockade. This work highlights that tumor-debris-fueled nanoreactors can benefit RFA by inhibiting tumor recurrence and preventing tumor metastasis., (© 2021. The Author(s).)

Passiflora edulis by-products (PFBP) are a rich source of polyphenols, of which piceatannol has gained special attention recently. However, there are few studies involving environmentally safe methods for obtaining extracts rich in piceatannol. This work aimed to concentrate piceatannol from defatted PFBP (d-PFBP) by means of pressurized liquid extraction (PLE) and conventional extraction, using the bio-based solvents selected with the Hansen solubility parameters approach. The relative energy distance ( Ra ) between solvent and solute was: Benzyl Alcohol (BnOH) < Ethyl Acetate (EtOAc) < Ethanol (EtOH) < EtOH:H 2 O. Nonetheless, EtOH presented the best selectivity for piceatannol. Multi-cycle PLE at 110 °C was able to concentrate piceatannol 2.4 times more than conventional extraction. PLE exhibited a dependence on kinetic parameters and temperature, which could be associated with hydrogen bonding forces and the dielectric constant of the solvents. The acetylcholinesterase (AChE) and lipoxygenase (LOX) IC 50 were 29.420 μg/mL and 27.682 μg/mL, respectively. The results reinforce the demand for processes to concentrate natural extracts from food by-products.

Many plants that are commonly used in folk medicine have multidirectional biological properties confirmed by scientific research. One of them is Aerva lanata (L.) Juss. (F. Amaranthaceae). It is widely used, but there are very few scientific data about its chemical composition and pharmacological activity. The aim of the present study was to investigate the chemical composition of phenolic acid (PA)-rich fractions isolated from methanolic extracts of A. lanata (L.) Juss. herb using the liquid/liquid extraction method and their potential antioxidant, anti-inflammatory, and anti-diabetic properties. The free PA fraction (FA), the PA fraction (FB) released after acid hydrolysis, and the PA fraction (FC) obtained after alkaline hydrolysis were analysed using liquid chromatography/electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). The phenolic profile of each sample showed a high concentration of PAs and their presence in A. lanata (L.) Juss. herb mainly in bound states. Thirteen compounds were detected and quantified in all samples, including some PAs that had not been previously detected in this plant species. Bioactivity assays of all fractions revealed high 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) (2.85 mM Trolox equivalents (TE)/g) and 2,2-azino-bis-3(ethylbenzthiazoline-6-sulphonic acid) (ABTS •+ ) (2.88 mM TE/g) scavenging activity. Fraction FB definitely exhibited not only the highest antiradical activity but also the strongest xanthine oxidase (XO) (EC 50 = 1.77 mg/mL) and lipoxygenase (LOX)(EC 50 = 1.88 mg/mL) inhibitory potential. The fraction had the best anti-diabetic properties, i.e., mild inhibition of α-amylase (EC 50 = 7.46 mg/mL) and strong inhibition of α-glucosidase (EC 50 = 0.30 mg/mL). The activities of all analysed samples were strongly related to the presence of PA compounds and the total PA content.

The five-membered heterocyclic group of pyrazoles/pyrazolines plays important role in drug discovery. Pyrazoles and pyrazolines present a wide range of biological activities. The synthesis of the pyrazolines and pyrazole derivatives was accomplished via the condensation of the appropriate substituted aldehydes and acetophenones, suitable chalcones and hydrazine hydrate in absolute ethanol in the presence of drops of glacial acetic acid. The compounds are obtained in good yields 68-99% and their structure was confirmed using IR, 1 H-NMR, 13 C-NMR and elemental analysis. The novel derivatives were studied in vitro for their antioxidant, anti-lipid peroxidation (AAPH) activities and inhibitory activity of lipoxygenase. Both classes strongly inhibit lipid peroxidation. Compound 2g was the most potent lipoxygenase inhibitor (IC 50 = 80 µM). The inhibition of the carrageenin-induced paw edema (CPE) and nociception was also determined, with compounds 2d and 2e being the most potent. Compound 2e inhibited nociception higher than 2d . Pyrazoline 2d was found to be active in a preliminary test, for the investigation of anti-adjuvant-induced disease (AID) activity. Pyrazoline derivatives were found to be more potent than pyrazoles. Docking studies of the most potent LOX inhibitor 2g highlight hydrophobic interactions with VAL126, PHE143, VAL520 and LYS526 and a halogen bond between the chlorine atom and ARG182.

Soybean lipoxygenase was immobilized on nanoporous rice husk silica particles by adsorption, and enzymatic parameters of the immobilized protein, including the efficiency of substrate binding and catalysis, kinetic and operational stability, and the kinetics of thermal inactivation, were investigated. The maximal adsorption efficiency of soybean lipoxygenase to the silica particles was 50%. The desorption kinetics of soybean lipoxygenase from the silica particles indicate that the silica-immobilized enzyme is more stable in an anionic buffer (sodium phosphate, pH 7.2) than in a cationic buffer (Tris-HCl, pH 7.2). The specific activity of immobilized lipoxygenase was 73% of the specific activity of soluble soybean lipoxygenase at a high concentration of substrate. The catalytic efficiency (k cat /K m ) and the Michaelis-Menten constant (K m ) of immobilized lipoxygenase were 21% and 49% of k cat /K m and K m of soluble soybean lipoxygenase, respectively, at a low concentration of substrate. The immobilized soybean lipoxygenase was relatively stable, as the enzyme specific activity was >90% of the initial activity after four assay cycles. The thermal stability of the immobilized lipoxygenase was higher than the thermal stability of soluble lipoxygenase, demonstrating 70% and 45% of its optimal specific activity, respectively, after incubation for 30 min at 45 °C. These results demonstrate that adsorption on nanoporous rice husk silica is a simple and rapid method for protein immobilization, and that adsorption may be a useful and facile method for the immobilization of many biologically important proteins of interest.

Karanja (Pongamia pinnata) is a medicinal tree used in the Indian traditional ayurvedic system for treating several ailments. The seeds contain a unique furano-flavonoid karanjin, which has shown to possess many medicinal properties. Its usage at the clinical level is affected due to poor solubility and absorption. In the present investigation, molecular modifications of karanjin were attempted and evaluated their effect on anti-inflammatory activity. Firstly, Karanja ketone was obtained from karanjin by hydrolysis, and it was converted into karanja ketone oxime. The oxime undergoes Beckmann rearrangement and cyclized to yield furano benzoxazole (karanja oxazole). The new derivatives were purified with >95% purity (HPLC) and spectrally characterized (HR-MS, FTIR, and NMR). Among the test compounds, karanja ketone oxime exhibited higher antioxidant activity with an IC 50 value of 360 µg/ml (DPPH). Soy lipoxygenase-1 (LOX-1) inhibitory activity of oxime was higher (IC 50  = 65.4 µM) than other compounds. Fluorescence studies showed that oxime had higher quenching capacity with a Q max of 76.3% and a binding constant of 0.9 × 10 5 M -1 for soy LOX-1. In-silico interaction studies showed that karanja ketone oxime had the least binding energy of -5.76 kcal/mol with LOX-1 by forming two hydrogen bonds with hydrophobic amino acids Leu 390 and Gly 392. The compounds were evaluated for their acute anti-inflammatory activity by the paw and ear edema in the rat model. Karanjin inhibits paw edema and ear edema by 34.13% and 51.13%, respectively, whereas the derivatives inhibited by 45-57 % and 70-76.8%. This study reports a rational approach to synthesize karanjin derivatives with considerable anti-inflammatory properties, both in-vitro and in-vivo., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: From the point of view of medicinal chemistry, compounds containing phenolic and pyrazolic moiety are significant since they are often constituents of bioactive compounds., Objective: The aims of this study were to synthesize pyrazole derivatives of medically relevant phenolic acids, confirm their structure, and evaluate their antioxidative and anti-LOX activities., Methods: Phenolic pyrazole derivatives were obtained, starting from esters of medically relevant phenolic acids. The structures of all obtained compounds were determined by NMR and IR spectroscopy, and UV-Vis spectrophotometry. In addition, the single-crystal X-ray diffraction was used. Pyrazole derivatives were tested for their in vitro antioxidative (DPPH assay), and lipoxygenase (LOX) inhibitory activities. Radical quenching mechanism was estimated using DFT and thermodynamic approach, while molecular docking was used to estimate the binding mode within the enzyme., Results: Pyrazole derivatives were obtained in high yields. The crystal structure of a new compound 3e was determined. Pyrazole derivative with catechol moiety 3d exhibited excellent radical scavenging activity, while compound 3b exhibited the best anti-LOX activity. Molecular docking study revealed that there is no direct interaction of any ligand with the active site of LOX-Ib, but pyrazoles 3a-e behave as inhibitors blocking the approach of linoleic acid to the active site., Conclusion: In this research, protocatechuic and vanillic acid pyrazole derivatives have been obtained for the first time. In vitro antioxidative assay suggests that pyrazole derivate of protocatechuic acid is a powerful radical scavenger, while anti-LOX assay indicates a pyrazole derivative with 4-hydroxyphenyl moiety., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Endogenous lipase and lipoxygenase play important roles in accelerating lipid oxidation. Polyphenols are a series of commonly used chemicals for preserving fish and seafood products, due to their positive inhibitory effects on lipid oxidation. However, the mechanism involved is still unknown. The inhibitory effects of chlorogenic acid (CGA) on lipase and lipoxygenase were investigated and explored with multi- spectroscopic and molecular docking approaches. Results showed that CGA could inhibit the activities of lipase and lipoxygenase with concentration increased in a highly dose-dependent manner. CGA quenched intrinsic fluorescence intensities of enzymes by static quenching and binding with CGA which led to changes in 3D structures of enzymes. Results of the molecular docking confirmed binding modes, binding sites and major interaction forces between CGA and enzymes, which reduced the corresponding activity. Thus, this study could provide basic mechanisms of the inhibitory effects of polyphenols on lipid oxidation during food preservation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

An overview of the biological properties of phycocyanin (PC) amply illustrates that it may not have any specific functional feature towards any system at which it may elicit a specific function, but for the molecular interactions. Nevertheless, based on existing evidences, it is hypothesized that PC has more than one functional target with the interacting systems; therefore, it has diversity of effects. The mechanism of PC action remains elusive of a comprehensive idea. The present investigation focuses on the pro inflammatory enzyme, lipoxygenase (LOX) inhibiting property of PC purified from Oscillatoria sp. Enzyme kinetics studies show that the molecular composite of PC is required for its inhibition shown on LOX. Isothermal titration calorimetric study proves that one molecule of PC interacts with two molecules of LOX. Molecular dynamics simulation study pertaining to PC-LOX interactions shows it to be appropriate as a model to give molecular mechanistic insight into the varied biological properties of PC, demonstrated elsewhere in experimental studies including animal model studies. It explains that the PC-LOX interaction is of a function-freezing, protein-protein interaction in nature. The wide spectrum of properties of PC might be due to its function as a powerful protein hub showing non-specific protein-protein interactions., Competing Interests: Declaration of competing interest The authors have declared no conflict of interests., (Copyright © 2020. Published by Elsevier B.V.)

North Pacific krill (Euphausia pacifica) contain 8R-hydroxy-eicosapentaenoic acid (8R-HEPE), 8R-hydroxy-eicosatetraenoic acid (8R-HETE) and 10R-hydroxy-docosahexaenoic acid (10R-HDHA). These findings indicate that E. pacifica must possess an R type lipoxygenase, although no such enzyme has been identified in krill. We analyzed E. pacifica cDNA sequence using next generation sequencing and identified two lipoxygenase genes (PK-LOX1 and 2). PK-LOX1 and PK-LOX2 encode proteins of 691 and 686 amino acids, respectively. Recombinant PK-LOX1 was generated in Sf9 cells using a baculovirus expression system. PK-LOX1 metabolizes eicosapentaenoic acid (EPA) to 8R-HEPE, arachidonic acid (ARA) to 8R-HETE and docosahexaenoic acid (DHA) to 10R-HDHA. Moreover, PK-LOX1 had higher activity for EPA than ARA and DHA. In addition, PK-LOX1 also metabolizes 17S-HDHA to 10R,17S-dihydroxy-docosahexaenoic acid (10R,17S-DiHDHA). PK-LOX1 is a novel lipoxygenase that acts as an 8R-lipoxygenase for EPA and 10R-lipoxygenase for DHA and 17S-HDHA. Our findings show PK-LOX1 facilitates the enzymatic production of hydroxy fatty acids, which are of value to the healthcare sector.

The effects of oxidation on the gastric digestion properties of soybean protein isolates (SPIs) in a model of lipoxygenase (LOX)-catalyzed linoleic acid (LA) oxidation system and the multiscale structural characterization of SPI hydrolysate were investigated. Results indicated that the feature of SPI hydrolysate is dependent upon the degree of oxidation. Pepsin hydrolysis caused a red shift in fluorescence intensity and a reduction in surface hydrophobicity and diminished the particle size of SPI hydrolysate during gastric digestion. Compared with the control, mild oxidation was beneficial to protein unfolding and gastric digestibility, as manifested by minimal molecular weight (MW) distribution >50 kDa (32.34%) and smaller peptide fragments under scanning electron microscopy. However, severe oxidation brought about 39.47% loss of free amino acids. It was interesting to find that glycinin was more vulnerable to pepsin hydrolysis after oxidation as compared to the native SPI. Overall, the moderately oxidized SPI appeared to be digested to a greater extent.

Omega-3 polyunsaturated fatty acids (PUFAs) have been known to have beneficial effects in the prevention of various diseases. Recently, it was identified that the bioactivities of omega-3 are related to lipid mediators, called pro-resolving lipid mediators (SPMs), converted from PUFAs, so they have attracted much attention as potential pharmaceutical targets. Here, we aimed to build an efficient production system composed of enzymatic and chemical catalysis that converts docosahexaenoic acid (DHA) into lipid mediators. The cyanobacterial lipoxygenase, named Osc-LOX, was identified and characterized, and the binding poses of enzyme and substrates were predicted by ligand docking simulation. DHA was converted into three lipid mediators, a 17S-hydroxy-DHA, a 7S,17S-dihydroxy-DHA (RvD5), and a 7S,15R-dihydroxy-16S,17S-epoxy-DPA (new type), by an enzymatic reaction and deoxygenation. Also, two lipid mediators, 7S,15R,16S,17S-tetrahydroxy-DPA (new type) and 7S,16R,17S-trihydroxy-DHA (RvD2), were generated from 7S,15R-dihydroxy-16S,17S-epoxy-DPA by a chemical reaction. Our study suggests that discovering new enzymes that have not been functionally characterized would be a powerful strategy for producing various lipid mediators. Also, this combination catalysis approach including biological and chemical reactions could be an effective production system for the manufacturing lipid mediators.

Lipoxygenases (LOXs) are potential treatment targets in a variety of inflammatory conditions. It is assumed that blocking the arachidonic acid (AA) metabolism via COX inhibition by either traditional NSAIDs or selective cyclooxygenase-2 (COX-2) inhibitors could lead to the generation of pro-inflammatory leukotrienes and lipoxins via the LOX pathway, partly accounting for the side effects seen with traditional NSAIDs and selective COX-2 inhibitors. To counter this, several LOX, phospholipase A 2 (PLA 2 ) inhibitors have been reported nowadays from natural sources. Cassia angustifolia (Vahl.) is a medicinal herb belonging to the family Leguminosae and their LOX inhibitory profiles are reported in this study. Results indicate that ethyl acetate extract of Cassia leaves could inhibit LOX. MS and IR data revealed the presence of aloe emodin (270.2 m/z) in the isolated fraction. Enzyme kinetics showed that aloe emodin inhibit Lipoxygenase competitively with an IC 50 of 29.49 μM. Interaction of aloe emodin with LOX was also studied using fluorescence quenching method. ITC results indicate that the interaction of LOX with aloe emodin is endothermic in nature with a stoichiometry of n = 3. In conclusion, anti-inflammatory property of the plant could be assigned to the presence of aloe emodin., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chronic inflammation is triggered by numerous diseases such as osteoarthritis, Crohn's disease and cancer. The control of the pro-inflammatory process can prevent, mitigate and/or inhibit the evolution of these diseases. Therefore, anti-inflammatory drugs have been studied as possible compounds to act in these diseases. This paper proposes a computational analysis of eugenol in relation to aspirin and diclofenac and analyzing the ADMET profile and interactions with COX-2 and 5-LOX enzymes, important enzymes in the signaling pathway of pro-inflammatory processes. Through the analysis of ADMET in silico, it was found that the pharmacokinetic results of eugenol are similar to NSAIDs, such as diclofenac and aspirin. Bioinformatics analysis using coupling tests showed that eugenol can bind to COX-2 and 5-LOX. These results corroborate with different findings in the literature that demonstrate anti-inflammatory activity with less gastric irritation, bleeding and ulcerogenic side effects of eugenol. The results of bioinformatics reinforce studies that try to propose eugenol as an anti-inflammatory compound that can act in the COX-2/5-LOX pathways, replacing some NSAIDs in different diseases.

The model of lipoxygenase-catalyzed linoleic acid (LA) oxidation was selected as representative of a lipid peroxidation system to investigate the effects of oxidative modification on soybean protein isolate (SPI) gel properties and in vitro gastric digestion. Fluorescence recovery after the photobleaching (FRAP) technique was applied to evaluate pepsin diffusion in the gel. The results showed that oxidative modification increased the gel hardness as well as brought about a compact and three-dimensional network structure, which consequently decreased the water mobility as manifest by lowering the relaxation time of T 2b and T 21 from 0.55 and 3.22 ms for the control to 0.32 and 2.42 ms for 7LA+LOX (addition of 7 mL of LA and LOX), respectively. It was interesting to note that pepsin diffusion was significant correlated ( p < 0.05) with T 2b and DH (degree of hydrolysis), indicating that water mobility might be a factor related to FITC-pepsin diffusion, which would ultimately influence the gel gastric digestion. Compared with native SPI, moderate oxidation can improve the digestibility of SPI gel by the summed effects of pepsin diffusion limitation, microstructure variation, and hydrolysis degree.

Hydrogen tunneling in enzymatic C-H activation requires a dynamical sampling among ground-state enzyme-substrate (E-S) conformations, which transiently generates a tunneling-ready state (TRS). The TRS is characterized by a hydrogen donor-acceptor distance (DAD) of 2.7 Å, ∼0.5 Å shorter than the dominant DAD of optimized ground states. Recently, a high-resolution, 13 C electron-nuclear double-resonance (ENDOR) approach was developed to characterize the ground-state structure of the complex of the linoleic acid (LA) substrate with soybean lipoxygenase (SLO). The resulting enzyme-substrate model revealed two ground-state conformers with different distances between the target C11 of LA and the catalytically active cofactor [Fe(III)-OH]: the active conformer " a ", with a van der Waals DAD of 3.1 Å between C11 and metal-bound hydroxide, and an inactive conformer " b ", with a distance that is almost 1 Å longer. Herein, the structure of the E-S complex is examined for a series of six variants in which subtle structural modifications of SLO have been introduced either at a hydrophobic side chain near the bound substrate or at a remote residue within a protein network whose flexibility influences hydrogen transfer. A remarkable correlation is found between the ENDOR-derived population of the active ground-state conformer a and the kinetically derived differential enthalpic barrier for D versus H transfer, Δ E a , with the latter increasing as the fraction of conformer a decreases. As proposed, Δ E a provides a "ruler" for the DAD within the TRS. ENDOR measurements further corroborate the previous identification of a dynamical network coupling the buried active site of SLO to the surface. This study shows that subtle imperfections within the initial ground-state structures of E-S complexes are accompanied by compromised geometries at the TRS.

Protein oxidation results in structural modification which affects its digestion. The objective of this work was to investigate the influence of lipoxygenases (LOX) catalyzed linoleic acid (LA) oxidation on the structure and in vitro gastric digests of soybean protein isolate (SPI). Fluorescence recovery after photobleaching (FRAP) was used to evaluate the relationship between pepsin diffusion and gastric digestion. Results indicated that oxidation induced carbonyl formation and loss of free sulfhydryl. Increased surface hydrophobicity and zeta-potential verified the protein unfolding and thus resulted in a small particle size and low fluorescence intensity. Fourier transform infrared spectroscopy (FTIR) showed that oxidation caused the increases in β-sheets mostly at the expense of α-helix and random coils. Fluorescein isothiocyanate (FITC)-pepsin in SPI solution modified with 3 mL LA showed a faster diffusion rate with 80.51 μm 2 /s as well as a higher DH value of 9.11%, showing that pepsin diffusivity might play an important role in protein gastric digestion.

Lipoxygenases (LOXs) are non-heme iron-containing dioxygenases involved in many developmental and stress-responsive processes in plants. However, little is known about the radish LOX gene family members and their functions in response to biotic and abiotic stresses. In this study, we completed a genome-wide analysis and expression profiling of RsLOX genes under abiotic and biotic stress conditions. We identified 11 RsLOX genes, which encoded conserved domains, and classified them in 9-LOX and 13-LOX categories according to their phylogenetic relationships. The characteristic structural features of 9-LOX and 13-LOX genes and the encoded protein domains as well as their evolution are presented herein. A qRT-PCR analysis of RsLOX expression levels in the roots under simulated drought, salinity, heat, and cold stresses, as well as in response to a Plasmodiophora brassicae infection, revealed three tandem-clustered RsLOX genes that are involved in responses to various environmental stresses via the jasmonic acid pathway. Our findings provide insights into the evolution and potential biological roles of RsLOXs related to the adaptation of radish to stress conditions.

Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first- and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific "binding network" that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O 2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate., (© 2019 Hu et al.)

This study aimed to investigate the effect of hydrogen peroxide (H 2 O 2 ) on membrane lipids metabolism and its relation to pulp breakdown development of longan fruit during postharvest storage. Compared to the control longans, H 2 O 2 -treated longans showed higher pulp breakdown index, cell membrane permeability, and activities of phospholipase D (PLD), lipase and lipoxygenase (LOX). Moreover, H 2 O 2 -treated longans maintained higher levels of pulp phosphatidic acid (PA) and saturated fatty acids (SFA). However, H 2 O 2 -treated longans exhibited lower levels of pulp phosphatidylcholine (PC), phosphatidylinositol (PI) and unsaturated fatty acids (USFA), lower index of unsaturated fatty acids (IUFA), and lower ratio of USFA to SFA (U/S). These findings demonstrated that H 2 O 2 caused the increased activities of enzymes involving in membrane lipids degradation and the accelerated decompositions of membrane USFA and phospholipids in longan pulp, which eventually triggered the destruction of the pulp cell membrane structure and the development of pulp breakdown in longans during storage., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (positions 317-334) that correlate well with trends in the corresponding enthalpic barriers for k cat . A model for this behavior posits that collisions between water and loop 317-334 initiate thermal activation at the protein surface that is then propagated 15-34 Å inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317-334 (Q322C) or on a control loop, 586-603 (S596C). Chemical modification of cysteines with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (Badan, BD) provides site-specific probes for the measurement of fluorescence relaxation lifetimes and Stokes shift decays as a function of temperature. Computational studies indicate that surface water structure is likely to be largely preserved in each sample. While both loops exhibit temperature-independent fluorescence relaxation lifetimes as do the Stokes shifts for S596C-BD, the activation enthalpy for the nanosecond solvent reorganization at Q322C-BD ( E a ( k solv ) = 2.8(0.9) kcal/mol)) approximates the enthalpy of activation for catalytic C-H activation ( E a ( k cat ) = 2.3(0.4) kcal/mol). This study establishes and validates the methodology for measuring rates of rapid local motions at the protein/solvent interface of SLO. These new findings, when combined with previously published correlations between protein motions and the rate-limiting hydride transfer in a thermophilic alcohol dehydrogenase, provide experimental evidence for thermally induced "protein quakes" as the origin of enthalpic barriers in catalysis.

The effect of elicitation with arachidonic and jasmonic acids on the production of phenolic compounds as well as the antioxidant and anti-inflammatory properties of phenolic extracts of wheatgrass was evaluated. The qualitative and quantitative analysis of phenolic compounds carried out with the UPLC-MS technique indicated that luteolin and apigenin derivatives were the dominant flavonoids, while ferulic acid derivatives and syringic acid were the main components in the phenolic acid fraction in the wheatgrass. No qualitative changes in the examined phenolic compounds were observed in the case of the control and elicited plants, while there was an increase in the content of some compounds. The antioxidant activity increased in the elicited samples (with the exception of reducing power) and this elevation was partially correlated with the increase in the polyphenol content in the studied plants. Elicitation with 0.01 µM arachidonic acid also caused improvement of potential anti-inflammatory properties of the wheatgrass., (Copyright © 2019. Published by Elsevier Ltd.)

Lipoxygenases from pathogenic fungi belong to the lipoxygenase family of enzymes, which catalyze C-H activation of polyunsaturated fatty acids to form a diverse set of cell-signaling hydroperoxides. While the lipoxygenase catalytic domains are structurally and functionally similar, these fungal enzymes are decorated with N-linked glycans. The impact of N-linked glycans on the structure and function of these enzymes remains largely unknown. One exemplary system is MoLOX, a lipoxygenase from the fungus Magnaporthe oryzae , that is emerging as an important target for the devastating rice blast disease. Herein, we demonstrate that hydrogen transfer, associated with C-H cleavage of the substrate linoleic acid by MoLOX, is rate-determining and occurs by a hydrogen tunneling mechanism. Using the differential enthalpic barrier for hydrogen and deuterium transfer, Δ E a , as a kinetic reporter of tunneling efficiency, a disproportionate increase in the activation energy for deuterium transfer is observed upon treatment of MoLOX with a peptide: N -glycosidase that cleaves N-linked carbohydrates from the protein. This increased Δ E a is consistent with an impairment of substrate positioning in the enzyme-substrate complex for both the tunneling ready state and the ground state. These results provide new insight into the functional consequences of N-linked glycosylation on lipoxygenase C-H activation and have important implications for MoLOX inhibitor design.

Accurate modeling of structural dynamics of proteins and their differentiation across different species can help us understand generic mechanisms of function shared by family members and the molecular basis of the specificity of individual members. We focused here on the family of lipoxygenases, enzymes that catalyze lipid oxidation, the mammalian and bacterial structures of which have been elucidated. We present a systematic method of approach for characterizing the sequence, structure, dynamics, and allosteric signaling properties of these enzymes using a combination of structure-based models and methods and bioinformatics tools applied to a data set of 88 structures. The analysis elucidates the signature dynamics of the lipoxygenase family and its differentiation among members, as well as key sites that enable its adaptation to specific substrate binding and allosteric activity.

Non-bullous congenital ichthyosiform erythroderma (NCIE) is characterized by skin scaling with erythema. In this study, two Pakistani families with NCIE are genetically characterized through Whole Exome and Sanger sequencing to identify molecular basis of the disease. We identified a nonsense homozygous c.2026C>T mutation of ALOXE3, causing premature termination of the eLOX3 protein (p.Q676X). In silico studies predicted impaired enzymatic activity of the premature truncated eLOX3, leading to abnormal synthesis of specific hepoxilin derivatives, essential for epidermal barrier formation. It is the first ever study reporting homozygotes of p.Q676X mutation in ethnically distinct two Pakistani families; otherwise, heterozygotes of the said mutation have been reported in South Asian population only. Hence, mutation seems to be region-specific and may be useful for molecular diagnosis of NCIE. Moreover, our findings should help in genetic counseling and career screening., (© 2018 Japanese Teratology Society.)

Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and trihydroxy fatty acids, which are biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are involved in pathogen-specific defense mechanisms against fungal infections. However, their quantitative biotransformation by plant enzymes has not been reported. A few bacteria produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis of plant oxylipins in bacteria have not been reported. In this study, we first report the biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9 S-lipoxygenase with and without epoxide hydrolase from the proteobacterium Myxococcus xanthus in recombinant Escherichia coli. Among the nine types of plant oxylipins, 12,13-epoxy-14-hydroxy- cis, cis-9,15-octadecadienoic acid was identified as a new compound by NMR analysis, and 9,10,11-hydroxy- cis, cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy- cis, cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This study shows that bioactive plant oxylipins can be produced by microbial enzymes.

Self-assembling amphipathic peptides (SAPs) have been used as stabilization tags to improve enzyme stability but do not function uniformly well with all target enzymes. Here, the key factors involved in SAPs stabilization were identified as the SAP length and linker length and flexibility, using S1 (AEAEAKAK) 2 as an originated SAP and polygalacturonate lyase (PGL) as model protein. Biochemical analysis demonstrated that SAPs could induce loose protein oligomerization via intermolecular hydrophobic interactions. Based on this mechanism, a comprehensive protein stabilization strategy was proposed, in which a library of stabilizing tags through random combination of different SAPs and linker peptides was developed to design the fusion composition while the sodium chloride (NaCl) was used to enhance the intermolecular hydrophobic interactions. By using the strategy, the PGL, lipoxygenase (LOX) and L-asparaginase exhibited 33.25-, 17.55- and 15.6-fold increases, respectively, in the t 1/2 value relative to that of the corresponding wild-type enzyme. The SAP library therefore shows great application potential in stability enhancement of enzymes/proteins., (Copyright © 2018. Published by Elsevier Inc.)

Soybean lipoxygenase (SLO) has served as a prototype for understanding the molecular origin of enzymatic rate accelerations. The double mutant (DM) L546A/L754A is considered a dramatic outlier, due to the unprecedented size and near temperature-independence of its primary kinetic isotope effect, low catalytic efficiency, and elevated enthalpy of activation. To uncover the physical basis of these features, we herein apply three structural probes: hydrogen-deuterium exchange mass spectrometry, room-temperature X-ray crystallography and EPR spectroscopy on four SLO variants (wild-type (WT) enzyme, DM, and the two parental single mutants, L546A and L754A). DM is found to incorporate features of each parent, with the perturbation at position 546 predominantly influencing thermally activated motions that connect the active site to a protein-solvent interface, while mutation at position 754 disrupts the ligand field and solvation near the cofactor iron. However, the expanded active site in DM leads to more active site water molecules and their associated hydrogen bond network, and the individual features from L546A and L754A alone cannot explain the aggregate kinetic properties for DM. Using recently published QM/MM-derived ground-state SLO-substrate complexes for WT and DM, together with the thorough structural analyses presented herein, we propose that the impairment of DM is the combined result of a repositioning of the reactive carbon of linoleic acid substrate with regard to both the iron cofactor and a catalytically linked dynamic region of protein.

Soybean lipoxygenase, recombinant rice allene oxide synthase-1 and rice allene oxide cyclase were covalently immobilized on nanoporous rice husk silica using two types of linkers: glutardialdehyde and polyethylene glycol. The immobilization efficiency achieved using glutardialdehyde-linked rice husk silica was higher than that achieved using polyethylene glycol-linked rice husk silica (50-92% and 25-50%, respectively). Immobilization on both types of matrices significantly decreased the specific activities of the immobilized enzymes. Solid-phase reaction yields of the enzymes were determined relative to the yields observed for the solution-phase reactions. Yields of the solid-phase reactions catalyzed by immobilized soybean lipoxygenase, rice allene oxide synthase-1, and rice allene oxide cyclase ranged from 50% to 230% and were dependent on both the enzymes and linkers used. Production of cis(+)-12-oxophytodienoic acid from α-linolenic acid by consecutive reactions using all three enzymes in a co-immobilization system resulted in 83.6% and 65.1% yields on glutardialdehyde-linked and epichlorohydrin-polyethylene glycol-linked rice husk silica, respectively. Our results suggest that immobilization of biosynthetic enzymes of the octadecanoid pathway on rice husk silica may be an efficient method for the in vitro production of oxylipins. Additionally, enzyme immobilizations on rice husk silica matrices may be more broadly applicable for producing physiologically important compounds in other biosynthetic pathways.