M. Haridas

X-ray structure of a galactose-specific lectin from Spatholobous parviflorous

A galactose-specific seed lectin from Spatholobous parviflorus (SPL) has been purified, crystallized and its X-ray structure solved. It is the first lectin purified and crystallized from the genus Spatholobus (family: Fabaceae). The crystals belong to the space group P1, with a=60.792 Å, b=60.998 Å, c=78.179 Å, α=78.68°, β=88.62°, γ=104.32°. The data were collected at 2.04 Å resolution under cryocondition, on a MAR image-plate detector system, mounted on a rotating anode X-ray generator. The coordinates of Dolichos biflorus lectin (1lu1) were successfully used for the structure solution by molecular replacement method. The primary structure of the SPL was not known earlier and it was unambiguously visible in the electron density. S. parviflorus lectin is a hetero-dimeric-tetramer with two alpha and two beta chains of 251 and 239 residues respectively. SPL has two metal ions, Ca(2+) and Mn(2+), bound to a loop region of each chain. The SPL monomers are in jelly roll form.

Inverted binding due to a minor structural change in berberine enhances its phospholipase A2 inhibitory effect

Biotransformation of berberine by Rhizopus oryzae leads to its demethylation, producing hydroxyl derivatives, as revealed by Fourier Transform Infra Red spectroscopy, Nuclear Magnetic Resonance and Electro Spray Ionization-Mass Spectrometric analyses. Surface Plasmon Resonance and enzyme kinetic studies showed that biotransformed derivatives of berberine had a higher inhibitory potential than berberine towards phospholipase A(2). X-ray crystal structures demonstrated that biotransformed berberine binds to PLA(2) in an entirely different, inverted orientation with respect to the binding of berberine. This study brings out the significance of biotransformation in generation of better drug-lead compounds.

Traditional fermentation of Ayurvedic medicine yields higher proinflammatory enzyme inhibition compared to wine-model product

This study investigated the process of preparing fermented medicines as prescribed in Ayurveda, the traditional Indian system of medicine. Berberine, an alkaloid, was used as a model compound. Berberine is the active constituent of Berberis aristata and is alleged to have anti-inflammatory effect. Biotransformation of berberine was studied by the phospholipase A2 assay in fermented products prepared in traditional and commercially available brewers yeast-induced environments. Sugar and alcohol levels were estimated to indicate the culmination of fermentation. It was confirmed that traditional fermentation biotransforms berberine to a greater extent than commercially available brewers yeast-induced fermentation. Therefore, fermentation induced by commercially available yeast is no substitute for the ethnopharmacological and traditional fermentation prescribed in the traditional Indian system of medicine.

Inhibitory activity of IAA and IBA against lipoxygenase: in silico and in vitro validation

Inhibition of leukotriene biosynthesis is considered to be one of the potential treatment strategies for controlling inflammation, respiratory diseases and many neurodegenerative disorders. Designing of specific functional inhibitors against Lipoxygenases (LOX) has got considerable attention due to its ability to block leukotriene biosynthesis. Molecular docking analysis of two indole derivatives such as indoleacetic acid (IAA) and indolebutyric acid (IBA) are reported here. Both compounds give glide scores better than that of protocatechuic acid and nitro catechol, the two known LOX inhibitors. From the enzyme kinetic analysis, it was revealed that IAA and IBA inhibit competitively. The IC50 values determined for both IAA and IBA were 42.98 μM and 17.82 μM, respectively. The binding free energy of these compounds was determined using isothermal titration calorimetric assay and was found to be − 6.12 kcal/mol for IAA and − 7.84 kcal/mol for IBA. From the analysis, it can be concluded that both IAA and IBA might be useful as anti-inflammatory agents.

Crystal structure of porcine pancreatic phospholipase A2 in complex with 2-methoxycyclohexa-2-5-diene-1,4-dione

Curcumin possess anti-inflammatory activity. In this study, the binding of curcumin with PLA2 was studied using X-ray crystallography. Since the electron density found at the active site did not match with curcumin, 2-methoxycyclohexa-2-5-diene-1,4-dione (MCW) (the photodegraded product of curcumin) was fitted in the unexplained electron density. To understand the binding mode of actual curcumin, molecular docking study was carried out. The crystallographic and docked structures were superimposed with respect to the ligand position and it was found that curcumin was binding in the hydrophobic cavity of PLA2 with a binding energy of−16.81 Kcal/mol. The binding mode was of such a nature that it prevented the entry of the substrate to the hydrophobic active site. This study indicates that curcumin can act as an inhibitor of PLA2. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:FLS:1.

Crystallization and preliminary X-ray diffraction analysis of a galactose-specific lectin from the seeds of Butea monosperma.

The galactose-specific lectin from the seeds of Butea monosperma has been crystallized by the hanging-drop vapour-diffusion technique. The crystals belonged to space group P1, with unit-cell parameters a = 78.45, b = 78.91, c = 101.85 Å, α = 74.30, β = 76.65, γ = 86.88°. X-ray diffraction data were collected to a resolution of 2.44 Å under cryoconditions (100 K) using a MAR image-plate detector system mounted on a rotating-anode X-ray generator. Molecular-replacement calculations carried out using the coordinates of several structures of legume lectins as search models indicate that the galactose-specific lectin from B. monosperma forms an octamer.

Metal Ion Coordination Essential for Specific Molecular Interactions of Butea monosperma Lectin: ITC and MD Simulation Studies

Crystal structure of Butea monosperma seed lectin (BML) was analyzed and the metal ion geometry identified. In order to understand the role of metal ions for the structural stability and ligand binding, studies of demetallized protein were carried out. Binding of different ligands like GalNAc, lactose, and galactose onto native and demetallized protein was studied by isothermal titration calorimetry as well as molecular simulation methods. Molecular dynamics was applied to the structure after removing the coordinates of metal ions, to identify the effect of demetallization in silico. Docking studies of different sugar molecules as well as the fungal α-amylase was carried out and compared the interactions in the native and apo states. It was found that metal ions are important for the ligand binding with increased affinity. However, their absence did not make any alteration to the secondary structure. Though the metal ions were not coordinated to the loops contacting the α-amylase, the absence of metal ions reduced the protein-protein binding strength due to long-range changes in irregular structures of the lectin.

Dioxygenase from Aspergillus fumigatus MC8: molecular modelling and in silico studies on enzyme-substrate interactions

Flavoenzymes have been extensively studied for their structural and mechanistic properties because they find potential application as industrial biocatalysts. They are attractive for biocatalysis because of the selectivity, controllability and efficiency of their reactions. Some of these enzymes catalyse the oxidative modification of protein substrates. Among them oxygenases (monoxoygenases and dioxygenases) are of special interest because they are highly entantio as well as regio-selective and can be used for oxyfunctionalisation. Dioxygenase enzymes catalyse oxygenation reactions in which both di-oxygen atoms are incorporated into the product. A dioxygenase enzyme purified from Aspergillus fumigatus MC8 was subjected to protein digestion followed by peptide sequencing. The sequence analysis of the peptide fragments resulted in identifying its match with that of an extracellular dioxygenase sequence from the same species of fungus existing in the protein database. The sequence was submitted to protein homology/analogy recognition engine online server for homology modelling and the 3D structure was predicted. Subsequently, the in silico studies of the enzyme–substrate (protein–ligand) interaction were carried out by using the method of molecular docking simulations wherein the modelled dioxygenase enzyme (protein) was docked with the substrates (ligands), catechin and epicatechin.

Novel lipoxygenase inhibitor, 1-ethenoxy-2-methylbenzene, from marine cyanobacteria Microcoleus chthonoplastes

Abstract Lipoxygenase (LOX) inhibitors are considered to be important anti-inflammatory agents as it can control many inflammatory responses to some extent. Even though the marine bio-systems are not well explored, they are considered to be one of the promising repositories for drug lead molecules against variety of diseases. In the present study a new LOX inhibitor compound, 1-ethenoxy-2-methylbenzene, reported first time from a living system, Microcoleus chthonoplastes, has been isolated by activity guided fractionation and further structurally characterised by techniques such as FTIR, NMR and LC MS/MS. Further enzyme kinetics, isothermal titration calorimetry and molecular docking methods were used in order to get a better understanding of enzyme–ligand interactions. This exploration suggests its worthiness as a lead molecule for the development of a better anti-inflammatory drug. Its high structure–activity resemblance to cuminaldehyde from cumin seeds, which is earlier reported as a LOX inhibitor, is also established.

Gallic acid binding to Spatholobus parviflorus lectin provides insight to its quaternary structure forming.

Therapeutic effects of gallic acid (GA) have already been extensively studied. However, its interaction with lectins has not gained much attention. It is of interest to validate the binding profile of GA with Spatholobus parviflorus seed lectin. A combination of Isothermal Titration Calorimetry (ITC), haemagglutination assay and molecular docking was applied on SPL-GA interaction. ITC results showed four binding sites, stoichiometry, n=4, irrespective of the ratio of SPL:GA taken for titration. Difference among the four binding sites of a single molecule of SPL with regard to GA binding kinetic parameters was consistently varying. Similarly, the glide scores obtained for GA in the four different binding clefts of SPL were also conformed to the ITC. The binding of GA on SPL without affecting its sugar binding property could be considered as a boon for glycobiological research. From the presented studies, it could be proposed that the SPL-GA interactions may facilitate drug delivery by specific targeting/attachment by profiling of cell-surface glycans, followed by controlled release of drugs.