Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

Strategic enzymatic enantioselective desymmetrization of prochiral cyclohexa-2,5-dienones.

Asymmetric desymmetrization through the selective reduction of one double bond of prochiral 2,5-cyclohexadienones is highly challenging. A novel method has been developed for synthesizing chiral cyclohexenones by employing an ene-reductase (Bacillus subtilis YqjM) enzyme that belongs to the OYE family. Our strategy demonstrates high substrate scope and enantioselectivity towards substrates containing all-carbon as well as heteroatom (O, N)-containing quaternary centers. The mechanistic studies (kH/D = ∼1.8) indicate that hydride transfer is probably the rate-limiting step. Mutation of several active site residues did not affect the stereochemical outcomes. This work provides a convenient way of synthesizing various enantioselective γ,γ-disubstituted cyclohexanones using enzymes.

Interaction of major saffron constituent safranal with trypsin: An experimental and computational investigation.

Trypsin is a serine protease, an important digestive enzyme that digests the proteins in the small intestine. In the present study, we have investigated the interaction of safranal, a major saffron metabolite, with trypsin using spectroscopic and molecular docking analyses. Fluorescence emission spectra of trypsin were largely affected by the inner filter effect from safranal; that's why these were corrected using the standard procedure. The corrected fluorescence spectra have shown that the safranal quenched the intrinsic fluorescence of trypsin with a blue shift in the wavelength of emission maximum, which revealed that the microenvironment of the fluorophore became more hydrophobic. There was approximately 1: 1 fair binding between them, which increased with a rise in temperature. The interaction was favored, principally, by hydrophobic forces, and there was an efficient energy transfer from the fluorophore to the safranal. Synchronous fluorescence spectra suggested that the tryptophan residues were the major ones taking part in the fluorescence quenching of trypsin. Safranal also influenced the secondary structure of trypsin and caused partial unfolding. Molecular Docking and the Molecular Dynamics simulation of the free and complexed trypsin was also carried out. Safranal formed a stable, non-covalent complex within the S2'-S5' subsite. Moreover, two nearby tyrosine residues (Tyr39 and Tyr151) stabilized safranal through π-π interactions. Additionally, the presence of safranal led to changes in the protein flexibility and compactness, which could indicate changes in the surrounding of tryptophan residues, impacting their fluorescence. Furthermore, a loss in compactness is in line with the partial unfolding observed experimentally. Thus, both experimental and computational studies were in good agreement with each other.

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Application of the relative molar sensitivity method using GC-FID to quantify safranal in saffron (Crocus sativus L.).

Safranal is one flavor component of saffron, which is used as a spice, food additive, and crude drug. In ISO3632, safranal is defined as the compound that contributes to the quality of saffron, and many quantitative determination methods for safranal have been reported. However, safranal is volatile and degrades easily during storage, and an analytical standard with an exact known purity is not commercially available, making it difficult to quantify accurately the content of safranal in saffron. Here, we developed a method for quantifying safranal using relative molar sensitivity (RMS), called the RMS method, using a GC-flame ionization detector (GC-FID). We determined the RMS of safranal to 1,4-bis(trimethylsilyl)benzene-d4, a certified reference material commercially available, by a combination of quantitative NMR and chromatography. Using two GC-FID instruments made by different manufacturers to evaluate inter-instrument effect, the resultant RMS was 0.770, and the inter-instrument difference was 0.6%. The test solution, with a known safranal concentration, was measured by the RMS method, with an accuracy of 99.4-101%, repeatability of 0.81%, and reproducibility of 0.81-1.3%. Given the ease of degradation, high volatility, and uncertain purity of safranal reagents, the RMS method is a more accurate quantification approach compared to the calibration curve method and methods based on absorption spectrophotometry. Moreover, our findings revealed that the GC-FID makeup gas affected the RMS and quantitative values.

Fungal biotransformation of carvone and camphor by Aspergillus flavus and investigation of cytotoxic activities of naturally obtained essential oils.

In this study, the biotransformation of carvone and camphor by Aspergillus flavus and the products were investigated. The biotransformation reaction of carvone by A. flavus resulted in the production of neodihydrocarveol, dihydrocarvone, 2-cyclohexene-1-one,2-methyl-5-(1-methylethenyl), limonene-1,2-diol, trans-p-mentha-1(7),8-dien-2-ol, p-menth-8(10)-ene-2,9-diol, and the biotransformation reaction of camphor resulted in the production of 2 -campholenic acid, 2-cyclohexene-1-one,2-hydroxy-4,4,6,6-tetramethyl, α-campholene aldehyde. The naturally produced essential oils by biotransformation of carvone and camphor were observed to be cytotoxic to breast cancer cells while no significant inhibition was seen in the healthy cell line. Additionally, biotransformation products had the highest inhibition (74%) against aflatoxin B1. The bioactivities of biotransformation products are promising, and they can be further investigated for their therapeutic potential as active agents.

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins.

Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible ß-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation.

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

The effect of salt stress on the production of apocarotenoids and the expression of genes related to their biosynthesis in saffron.

Saffron stigmas are widely used as food additives and as traditional medicine in Iran and many other countries. The unique taste, flavor and pharmaceutical properties of saffron stigmas are due to the presence of three apocarotenoids secondary metabolites crocin, picrocrocin and safranal. There is limited knowledge about the effect of environmental stresses on the metabolism of apocarotenoids in saffron. We analyzed the content of crocin and picrocrocin and the expression of key genes of apocarotenoid biosynthesis pathways (CsCCD2, CsCCD4, CsUGT2, CsCHY-ß and CsLCYB) in saffron plants exposed to moderate (90 mM) and high (150 mM) salt (NaCl) concentrations. Measuring ion concentrations in leaves showed an increased accumulation of Na+ and decreased uptake of K+ in salt treated compared to control plants indicating an effective salt stress. HPLC analysis of apocarotenoids revealed that crocin production was significantly halted (P < 0.05) with increasing salt concentration while picrocrocin level did not change with moderate salt but significantly dropped by high salt concentration. Real-time PCR analysis revealed a progressive decrease in transcript levels of CsUGT2 and CsLCYB genes with increasing salt concentration (P < 0.05). The expression of CsCCD2 and CsCHY-ß tolerated moderate salt concentration but significantly downregulated with high salt concentration. CsCCD4 however responded differently to salt concentration being decreased with moderate salt but increased at higher salt concentration. Our result suggested that salt stress had an adverse effect on the production of saffron apocarotenoids and it is likely influencing the quality of saffron stigma produced.

Characterization of a 3-hydroxyanthranilic acid 6-hydroxylase involved in paulomycin biosynthesis.

Paulomycins (PAUs) refer to a group of glycosylated antibiotics with attractive antibacterial activities against Gram-positive bacteria. They contain a special ring A moiety that is prone to dehydrate between C-4 and C-5 to a quinone-type form at acidic condition, which will reduce the antibacterial activities of PAUs significantly. Elucidation of the biosynthetic mechanism of the ring A moiety may facilitate its structure modifications by combinatorial biosynthesis to generate PAU analogues with enhanced bioactivity or stability. Previous studies showed that the ring A moiety is derived from chorismate, which is converted to 3-hydroxyanthranilic acid (3-HAA) by a 2-amino-2-deoxyisochorismate (ADIC) synthase, a 2,3-dihydro-3-hydroxyanthranilic acid (DHHA) synthase, and a DHHA dehydrogenase. Unfortunately, little is known about the conversion process from 3-HAA to the highly decorated ring A moiety of PAUs. In this work, we characterized Pau17 as an unprecedented 3-HAA 6-hydroxylase responsible for the conversion of 3-HAA to 3,6-DHAA by in vivo and in vitro studies, pushing one step forward toward elucidating the biosynthetic mechanism of the ring A moiety of PAUs.

Impact of two different dehydration methods on saffron quality, concerning the prevalence of Saffron latent virus (SaLV) in Iran.

The dehydration process is a prerequisite to preserve saffron for a long time. According to this process, saffron shows differences in the main compounds responsible for its quality (colour, taste, aroma, and flavonol content). At present, the freeze-drying method obtains dried products with the highest quality. Viruses can modify the physiology and metabolism of plants, being able to affect the activities of several enzymes. For this reason, the main compounds of saffron have been analyzed under two different dehydrating processes, freeze-drying and dark-drying, considering their infection status with the Saffron latent virus (SaLV). Results showed that the picrocrocin and safranal content enables to differ dark-dried samples from freeze-dried ones. Besides, the kaempferol-3-O-sophoroside-7-O-glucoside content allows differentiating between SaLV-infected (SaLV+) and uninfected (SaLV-) saffron samples. Moreover, our data suggest that the freeze-drying would decrease crocins content, and dark-drying can nullify the adverse effect of SaLV on crocins content.

Efficient microbial resolution of racemic methyl 3-cyclohexene-1-carboxylate as chiral precursor of Edoxaban by newly identified Acinetobacter sp. JNU9335.

Optically active 3-cyclohexene-1-carboxylic acid (CHCA) derivatives are important pharmaceutical intermediates. Due to the special rotatable structure, enantioselective preparation of chiral CHCA is hard to achieve. To identify efficient and enantioselective hydrolases for the biosynthesis of CHCA from methyl 3-cyclohexene-1-carboxylate (CHCM), target-oriented screening from soil samples and gene mining from genome database were explored. All putative hydrolases attempted displayed low enantioselectivity. A hydrolase-producing strain JNU9335 was successfully identified with relatively high enantioselectivity, and was designated as a strain of Acinetobacter sp. according to 16S rDNA sequence and phylogenetic analysis. After optimization, strain JNU9335 could produce 233 U·L‒1 hydrolase with E value of 21. Isooctane/aqueous biphasic system is favorable for the enzymatic resolution of CHCM, the E value of JNU9335 could further be increased to 36. The newly identified JNU9335 could tolerate as high as 1.0 M CHCM, producing (S)-CHCM with ees of 99.6% and isolation yield of 34.7%. This study provides an efficient biocatalyst for the preparation of chiral 3-cyclohexene-1-carboxylic acid derivatives.

Enzymatic epoxidation of cyclohexene by peroxidase immobilization on a textile and an adapted reactor design.

A textile-based reaction system for new peroxidase reactions in non-native media was implemented. The epoxidation of cyclohexene by the commercial peroxidase MaxiBright® was realized with the textile-immobilized enzyme in an adapted liquid-liquid two-phase reactor. A commercially available polyester felt was used as low-price carrier and functionalized with polyvinyl amine. The covalent immobilization with glutardialdehyde lead to an enzyme loading of 0.10 genzyme/gtextile. The textile-based peroxidase shows a high activity retention in the presence of organic media. This catalyst is shown to enable the epoxidation of cyclohexene in various solvents as well as under neat conditions. A model reactor was produced by 3D printing which places the textile catalyst at the interphase between the liquid reaction phase and the product extracting solvent.

Studies on the Catalytic Promiscuity of Limonene Epoxide Hydrolases in the Non-hydrolytic Ring Opening of 1,2-Epoxides.

The non-hydrolytic ring opening of 1,2-epoxides in the presence of limonene epoxide hydrolases (LEHs) and different nucleophiles has been investigated. Lyophilized, wild-type LEHs were tested in selected water-saturated organic solvents in the presence of cyclohexene oxide as substrate and different alcohols, thiols and primary amines as nucleophiles. Although the LEHs retained an appreciable catalytic activity under different reaction conditions, formation of the desired 1,2-substituted cyclohexanols was not observed. Alternatively, LEH variants incapable of performing the hydrolytic reaction were generated by site-directed mutagenesis and tested in aqueous media in the presence of different water-soluble nucleophiles and cyclohexene oxide. Under defined reaction conditions, an acceleration of up to about threefold of the spontaneous reaction rate was observed in the presence of sodium azide and potassium thiocyanate as nucleophiles.

In Silico Investigation of Bitter Hop-Derived Compounds and Their Cognate Bitter Taste Receptors.

The typical bitter taste of beer is caused by adding hops (Humulus lupulus L.) during the wort boiling process. The bitter taste of hop-derived compounds was found to be mediated by three bitter taste receptors: TAS2R1, TAS2R14, and TAS2R40. In this work, structural bioinformatics analyses were used to characterize the binding modes of trans-isocohumulone, trans-isohumulone, trans-isoadhumulone, cis-isocohumulone, cis-isohumulone, cis-isoadhumulone, cohumulone, humulone, adhumulone, and 8-prenylnaringenin into the orthosteric binding site of their cognate receptors. A conserved asparagine in transmembrane 3 was found to be essential for the recognition of hop-derived compounds, whereas the surrounding residues in the binding site of the three receptors encode the ligand specificity. Hop-derived compounds are renowned bioactive molecules and are considered as potential hit molecules for drug discovery to treat metabolic diseases. A chemoinformatics analysis revealed that hop-derived compounds cluster in a different region of the chemical space compared to known bitter food-derived compounds, pinpointing hop-derived compounds as a very peculiar class of bitter compounds.

Arogenate dehydratases can modulate the levels of phenylacetic acid in Arabidopsis.

Phenylacetic acid (PAA) is one type of natural auxin and widely exists in plants. Previous biochemical studies demonstrate that PAA in plants is synthesized from phenylalanine (Phe) via phenylpyruvate (PPA), but the PAA biosynthetic genes and its regulation remain unknown. In this article, we show that the AROGENATE DEHYDRATASE (ADT) family, which catalyzes the conversion of arogenate to Phe, can modulate the levels of PAA in Arabidopsis. We found that overexpression of ADT4 or ADT5 remarkably increased the amounts of PAA. Due to an increase in PAA levels, ADT4ox and ADT5ox plants can partially restore the auxin-deficient phenotypes caused by treatments with an inhibitor of the biosynthesis of indole-3-acetic acid (IAA), a main auxin in plants. In contrast, the levels of PAA were significantly reduced in adt multiple knockout mutants. Moreover, the levels of PPA are substantially increased in ADT4 or ADT5 overexpression plants but reduced in adt multiple knockout mutants, suggesting that PPA is a key intermediate of PAA biosynthesis. These results provide an evidence that members of the ADT family of Arabidopsis can modulate PAA level via the PPA-dependent pathway.

A Validamycin Shunt Pathway for Valienamine Synthesis in Engineered Streptomyces hygroscopicus 5008.

Valienamine is the key functional component of many natural glycosidase inhibitors, including the crop protectant validamycin A and the clinical antidiabetic agent acarbose. Due to its important biomedical activity, it is also the prominent lead compound for the exploration of therapeutic agents, such as the stronger α-glucosidase inhibitor voglibose. Currently, the main route for obtaining valienamine is a multistep biosynthetic process involving the synthesis and degradation of validamycin A. Here, we established an alternative, vastly simplified shunt pathway for the direct synthesis of valienamine based on an envisioned non-natural transamination in the validamycin A producer Streptomyces hygroscopicus 5008. We first identified candidate aminotransferases for the non-natural ketone substrate valienone and conducted molecular evolution in vitro. The WecE enzyme from Escherichia coli was verified to complete the envisioned step with >99.9% enantiomeric excess and was further engineered to produce a 32.6-fold more active mutant, VarB, through protein evolution. Subsequently, two copies of VarB were introduced into the host, and the new shunt pathway produced 0.52 mg/L valienamine after a 96-h fermentation. Our study thus illustrates a dramatically simplified alternative shunt pathway for valienamine production and introduces a promising foundational platform for increasing the production of valienamine and its valuable N-modified derivatives for use in pharmaceutical applications.

Simultaneous measurement of perillyl alcohol and its metabolite perillic acid in plasma and lung after inhalational administration in Wistar rats.

The inhalational administration of drugs is a practical and non-invasive approach with the potential to reduce side effects and with a quick onset of therapeutic activity. Perillyl alcohol (POH) is a monoterpene with antitumor activity that currently is undergoing clinical evaluation as an inhalational anticancer agent. A detection method was developed that will be applicable to pharmacokinetic studies of not only POH, but also its longer-lived main metabolite, perillic acid (PA), in lung tissue and plasma after inhalational delivery. The anticancer activity of POH was investigated in vitro with the use of various lung cancer cell lines. Toxicity was established by a standard MTT assay, and apoptosis markers were analyzed by Western blot. For the detection of POH and PA in lungs and plasma, albino Wistar rats were used that were exposed to POH inhalation. Tissues were subjected to chromatographic separation on an Agilent Zorbax Eclipse XDB C18 column, followed by detection of absorption in the ultraviolet (UV) range. In vitro, POH exerted cytotoxic activity against six different lung tumor cell lines, and apoptotic cell death was indicated by induction of active caspase 3 and cleavage of poly (ADP-ribose) polymerase 1 (PARP1). These results demonstrate that inhalational delivery of POH results in effective biodistribution and metabolism of POH in the systemic circulation. In addition, our study introduces a simple, rapid HPLC-UV method with high accuracy for simultaneous detection of POH and its metabolite PA in plasma, and for sensitive detection of PA in lung tissue, which should prove useful for applications in clinical studies.

Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory.

BACKGROUND: Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. RESULTS: α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. CONCLUSIONS: An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.

Natural compound safranal driven inhibition and dis-aggregation of α-synuclein fibrils.

Self-assembly of α-synuclein (α-Syn) is linked with a variety of neurodegenerative diseases collectively called as α-synucleiopathies. Therefore, discovering suitable inhibitors for this self-association process of α-Syn is a subject of intense research. In this background, we have demonstrated here that the natural compound, Safranal, delays/inhibits α-Syn fibrillation/aggregation, and we have also characterized its mode of action. The α-Syn fibrillation/aggregation kinetics studies in combination with TEM studies demonstrated that Safranal effectively inhibits α-Syn fibrillation/aggregation. NMR studies revealed that Safranal binds with α-Syn and stabilizes the monomeric protein. ANS fluorescence and CD measurements indicated that Safranal binds to the hydrophobic residues of the protein and causes delay in the formation of ß-sheet rich structures which are crucial for the fibrillation to occur. The results obtained from fluorescence quenching, NMR and ANS binding assays, when analysed taking into consideration the molecular structure of Safranal provide valuable insights into the mechanism of inhibition of α-Syn fibrillation/aggregation. We infer that inhibition of α-Syn fibrillation/aggregation is primarily driven by hydrophobic interactions between Safranal and the protein. Further, Safranal is also seen to dis-aggregates pre-formed α-Syn fibrils. These findings implicate that Safranal could become a potent therapeutic intervention in Parkinson's disease and other protein aggregation related disorders.