Impact of N-Glycosylation on Protein Structure and Dynamics Linked to Enzymatic C-H Activation in the M. oryzae Lipoxygenase.

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, MoLOX. 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 MoLOX 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 MoLOX 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.

13C Electron Nuclear Double Resonance Spectroscopy-Guided Molecular Dynamics Computations Reveal the Structure of the Enzyme-Substrate Complex of an Active, N-Linked Glycosylated Lipoxygenase.

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 13C/1H 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 Mn2+ ion as a spin probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear Mn2+ metallocenters. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from rice blast pathogen Magnaporthe oryzae, MoLOX complexed with LA, as obtained through the 13C/1H 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 MoLOX-LA complex derived in this fashion is 3.4 ± 0.1 Å. The difference of the MoLOX-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 MoLOX 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 MoLOX inhibitors, and highlight the robustness of the ENDOR-guided MD approach to describe LOX-substrate structures.

Pseudophosphorylation of Arabidopsis jasmonate biosynthesis enzyme lipoxygenase 2 via mutation of Ser600 inhibits enzyme activity.

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 Ser600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2WT). AtLOX2WT 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 Ser600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2S600D and Atlox2S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2S600A 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 Ser600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser600 is located at a critical position within the AtLOX2 structure and highlights how Ser600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.

Inhibitory effects of ultrasonic and rosmarinic acid on lipid oxidation and lipoxygenase in large yellow croaker during cold storage.

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.

Dynamical activation of function in metalloenzymes.

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 Ea 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 Ea values measured for kcat . 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.

Interactions of potato-derived and human recombinant 5-lipoxygenase with sec-O-glucosylhamaudol by multi-spectroscopy and molecular docking.

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.

Mimicking Elementary Reactions of Manganese Lipoxygenase Using Mn-hydroxo and Mn-alkylperoxo Complexes.

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 MnIII-hydroxo center initiates substrate oxidation. In some proposed mechanisms, the active-site MnIII-hydroxo complex is regenerated by the reaction of a MnIII-alkylperoxo intermediate with water by a ligand substitution reaction. In a recent study, we described a pair of MnIII-hydroxo and MnIII-alkylperoxo complexes supported by the same amide-containing pentadentate ligand (6Medpaq). In this present work, we describe the reaction of the MnIII-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 MnIII-hydroxo complex [MnIII(OH)(6Medpaq)]+ oxidizes TEMPOH (2,2'-6,6'-tetramethylpiperidine-1-ol) faster than the majority of previously reported MnIII-hydroxo complexes. Using a combination of cyclic voltammetry and electronic structure computations, we demonstrate that the weak MnIII-N(pyridine) bonds lead to a higher MnIII/II reduction potential, increasing the driving force for substrate oxidation reactions and accounting for the faster reaction rate. In addition, we demonstrate that the MnIII-alkylperoxo complex [MnIII(OOtBu)(6Medpaq)]+ reacts with water to obtain the corresponding MnIII-hydroxo species, thus mimicking the ligand substitution step proposed for MnLOX.

Controlling the Reactivity of a Metal-Hydroxo Adduct with a Hydrogen Bond.

The enzymes manganese lipoxygenase (MnLOX) and manganese superoxide dismutase (MnSOD) utilize mononuclear Mn centers to effect their catalytic reactions. In the oxidized MnIII 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 MnIII-hydroxo complexes that differ by a single functional group. One of these complexes, [MnIII(OH)(PaPy2N)]+, contains a naphthyridinyl moiety capable of forming an intramolecular hydrogen bond with the hydroxo ligand. The second complex, [MnIII(OH)(PaPy2Q)]+, 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 [MnIII(OH)(PaPy2N)]+, consistent with a hydrogen bond. Kinetic studies using a variety of substrates with activated O-H bonds, revealed that [MnIII(OH)(PaPy2N)]+ is far more reactive than [MnIII(OH)(PaPy2Q)]+, 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 [MnII(OH2)(PaPy2N)]+ product. Thus, the differences in reactivity between [MnIII(OH)(PaPy2Q)]+ and [MnIII(OH)(PaPy2N)]+ 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.

Combining Structural with Functional Model Properties in Iron Synthetic Analogue Complexes for the Active Site in Rabbit Lipoxygenase.

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.

Design of Multifaceted Antioxidants: Shifting towards Anti-Inflammatory and Antihyperlipidemic Activity.

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.

Pyrazoles and Pyrazolines as Anti-Inflammatory Agents.

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, 1H-NMR, 13C-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 (IC50 = 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.

Tumor-killing nanoreactors fueled by tumor debris can enhance radiofrequency ablation therapy and boost antitumor immune responses.

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 CaCO3-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.

Selective Extraction of Piceatannol from Passiflora edulis by-Products: Application of HSPs Strategy and Inhibition of Neurodegenerative Enzymes.

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:H2O. 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) IC50 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.

Antioxidant, Anti-Inflammatory, and Anti-Diabetic Activity of Phenolic Acids Fractions Obtained from Aerva lanata (L.) Juss.

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) (EC50 = 1.77 mg/mL) and lipoxygenase (LOX)(EC50 = 1.88 mg/mL) inhibitory potential. The fraction had the best anti-diabetic properties, i.e., mild inhibition of α-amylase (EC50 = 7.46 mg/mL) and strong inhibition of α-glucosidase (EC50 = 0.30 mg/mL). The activities of all analysed samples were strongly related to the presence of PA compounds and the total PA content.

A 13-Lipoxygenase, GhLOX2, positively regulates cotton tolerance against Verticillium dahliae through JA-mediated pathway.

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 H2O2 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.

Bioactive stigmastadienone from Isodon rugosus as potential anticholinesterase, α-glucosidase and COX/LOX inhibitor: In-vitro and molecular docking studies.

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 (H2O2) 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 (IC50) values of 13.52 and 11.53 µg/ml for acetyl and butyrylcholinesterase respectively. The observed IC50 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 IC50 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 IC50 values of 4.72 and 3.36 µg/ml respectively. The standard drugs IC50 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.

Oxygenation of endocannabinoids by mammalian lipoxygenase isoforms.

Endocannabinoids, such as anandamide (ANA) and 2-arachidonoylglycerol (2AG), are lipid-signaling molecules that can be oxidized by lipid-peroxidizing enzymes, and this oxidation alters the bioactivity of these lipid mediators. Here, under strictly comparable experimental conditions, we explored whether ANA and 2AG function as substrates for four human (ALOX15, ALOX15B, ALOX12, ALOX5) and three mice Alox isoforms (Alox15, Alox12, Alox5) and compared the rates of product formation with those of arachidonic acid oxygenation. Except for ALOX5, the two endocannabinoids were more efficiently oxygenated than arachidonic acid by human ALOX isoforms. Mice Alox15 oxygenated ANA more efficiently than arachidonic acid, but the other mice Alox isoforms exhibited reduced reaction rates for endocannabinoid conversion. Like its human ortholog, mice Alox5 did not oxygenate ANA, but the formation of 5-HETE-containing 2AG derivatives was observed for this enzyme. 1AG and 2AG were similarly effective substrates for human ALOX isoforms. Molecular docking studies, the pattern of oxygenation products, and site-directed mutagenesis experiments suggested a similar substrate alignment of arachidonic acid and endocannabinoids at the active site of ALOX15 orthologs. The product specificity of arachidonic acid oxygenation was conserved for endocannabinoid metabolization, and the triad concept describing the molecular basis for the reaction specificity of ALOX15 orthologs is applicable for endocannabinoid oxygenation. Taken together, these data indicate that, except for ALOX5 orthologs, endocannabinoids are suitable substrates for most mammalian ALOX isoforms.

Immobilization of Soybean Lipoxygenase on Nanoporous Rice Husk Silica by Adsorption: Retention of Enzyme Function and Catalytic Potential.

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 (kcat/Km) and the Michaelis-Menten constant (Km) of immobilized lipoxygenase were 21% and 49% of kcat/Km and Km 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.

Synthesis, characterization and anti-inflammatory properties of karanjin (Pongamia pinnata seed) and its derivatives.

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 IC50 value of 360 µg/ml (DPPH). Soy lipoxygenase-1 (LOX-1) inhibitory activity of oxime was higher (IC50 = 65.4 µM) than other compounds. Fluorescence studies showed that oxime had higher quenching capacity with a Qmax of 76.3% and a binding constant of 0.9 × 105 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.

Identification of a novel enzyme from E. pacifica that acts as an eicosapentaenoic 8R-LOX and docosahexaenoic 10R-LOX.

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.