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VIT University

Department of Social Sciences

A novel plant protein isolated from the underground bulbs of Scadoxus multiflorus, xylanase and α-amylase inhibitor protein (XAIP), inhibits two structurally and functionally unrelated enzymes: xylanase and α-amylase. The mature protein contains 272 amino acid residues which show sequence identities of 48% to the plant chitinase hevamine and 36% to xylanase inhibitor protein-I, a double-headed inhibitor of GH10 and GH11 xylanases. However, unlike hevamine, it is enzymatically inactive and, unlike xylanase inhibitor protein-I, it inhibits two functionally different classes of enzyme. The crystal structure of XAIP has been determined at 2.0 ̊ resolution and refined to Rcryst and Rfree factors of 15.2% and 18.6%, respectively. The polypeptide chain of XAIP adopts a modified triosephosphate isomerase barrel fold with eight β-strands in the inner circle and nine α-helices forming the outer ring. The structure contains three cis peptide bonds: Gly33-Phe34, Tyr159-Pro160 and Trp253-Asp254. Although hevamine has a long accessible carbohydrate-binding channel, in XAIP this channel is almost completely filled with the side-chains of residues Phe13, Pro77, Lys78 and Trp253. Solution studies indicate that XAIP inhibits GH11 family xylanases and GH13 family α-amylases through two independent binding sites located on opposite surfaces of the protein. Comparison of the structure of XAIP with that of xylanase inhibitor protein-I, and docking studies, suggest that loops α3-β4 and α4-β5 may be involved in the binding of GH11 xylanase, and that helix α7 and loop β6-α6 are suitable for the interaction with α-amylase. © 2010 FEBS.

School of Social Sciences & Languages

Vellore Campus

World University root node

Centre for Bio-Separation and Technology

Fulltext

FEBS Journal

Crystal structure determination and inhibition studies of a novel xylanase and α-amylase inhibitor protein (XAIP) from Scadoxus multiflorus

Background. Plants produce a wide range of proteinaceous inhibitors to protect themselves against hydrolytic enzymes. Recently a novel protein XAIP belonging to a new sub-family (GH18C) was reported to inhibit two structurally unrelated enzymes xylanase GH11 and -amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of -amylase GH13. A new form of XAIP (XAIP-II) that inhibits -amylase GH13 with a greater potency than that of XAIP and xylanase GH11 with a lower potency than that of XAIP, has been identified in the extracts of underground bulbs of Scadoxus multiflorus. This kind of occurrence of isoforms of inhibitor proteins is a rare observation and offers new opportunities for understanding the principles of protein engineering by nature. Results. In order to determine the structural basis of the enhanced potency of XAIP-II against -amylase GH13 and its reduced potency against xylanase GH11 as compared to that of XAIP, we have purified XAIP-II to homogeneity and obtained its complete amino acid sequence using cloning procedure. It has been crystallized with 0.1 M ammonium sulphate as the precipitating agent and the three-dimensional structure has been determined at 1.2 resolution. The binding studies of XAIP-II with xylanase GH11 and -amylase GH13 have been carried out with surface plasmon resonance (SPR). Conclusion. The structure determination revealed that XAIP-II adopts the well known TIM barrel fold. The xylanase GH11 binding site in XAIP-II is formed mainly with loop 3-3 (residues, 102 - 118) which has acquired a stereochemically less favorable conformation for binding to xylanase GH11 because of the addition of an extra residue, Ala105 and due to replacements of two important residues, His106 and Asn109 by Thr107 and Ser110. On the other hand, the -amylase binding site, which consists of -helices 6 (residues, 193 - 206), 7 (residues, 230 - 243) and loop 6-6 (residues, 180 - 192) adopts a stereochemically more favorable conformation due to replacements of residues, Ser190, Gly191 and Glu194 by Ala191, Ser192 and Ser195 respectively in -helix 6, Glu231 and His236 by Thr232 and Ser237 respectively in -helix 7. As a result, XAIP-II binds to xylanase GH11 less favorably while it interacts more strongly with -amylase GH13 as compared to XAIP. These observations correlate well with the values of 4.2 × 10 -6 M and 3.4 × 10-8 M for the dissociation constants of XAIP-II with xylanase GH11 and -amylase GH13 respectively and those of 4.5 × 10-7 M and 3.6 × 10-6 M of XAIP with xylanase GH11 and -amylase GH13 respectively. © 2010 Kumar et al; licensee BioMed Central Ltd.

BMC Structural Biology

Modulation of inhibitory activity of xylanase - α-amylase inhibitor protein (XAIP): Binding studies and crystal structure determination of XAIP- II from Scadoxus multiflorus at 1.2 Å resolution

Glutathione S-transferase P1 (GSTP1) is a member of the GST superfamily, which has well-established multiple roles in various infectious and parasitic diseases. The genetic regulation of GSTP1 has been extensively studied. Thus, its biological significance and disease association prompted us to investigate the role of GSTP1 polymorphisms in Plasmodium-mediated pathogenesis in infected humans. The genotypic distribution of Ile105Val in Plasmodium vivax infection was observed to be significant and strongly associated (OR = 4.5) with the progression of pathology, whereas in P. falciparum infection no significant association was observed compared to healthy subjects. Interestingly, we observed significant elevation of GST in vivax infection, with both genotypes Ile105Val and Val105Val, compared to healthy subjects, whereas in P. falciparum infection we found marginally elevated GST levels of mutated genotypes but significantly depleted compared to healthy subjects. Further, during vivax and falciparum infection overall significant elevations of glutathione, glutathione peroxidase, and GST levels were observed. Expression of both GSTP1 mRNA and protein was significantly upregulated during vivax infection compared to falciparum infection and both were significantly upregulated compared to the levels in healthy subjects as well. These studies suggest that GSTP1 polymorphism is involved in the pathogenesis of malaria and it may serve as a valuable molecular marker, possessing a promising rationale for diagnostic potential in assessing disease progression during clinical malaria. © 2010 Elsevier Inc.

Free Radical Biology and Medicine

Polymorphism in glutathione S-transferase P1 is associated with susceptibility to Plasmodium vivax malaria compared to P. falciparum and upregulates the GST level during malarial infection

A novel antifungal protein, amaryllin, has been isolated from the underground bulbs of Amaryllis belladonna, purified to homogeneity and crystallized. The protein was extracted using ammonium sulfate fractionation. The purified protein samples indicated a molecular weight of 15 kDa on SDS-PAGE. The protein showed antifungal activity against Aspergillus flavus and Fusarium oxysporum. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation and did not show significant sequence identity to any known protein. The protein was crystallized using the hanging-drop vapour-diffusion method with 30% PEG 8000 as precipitating agent. The crystals diffracted to 2.7 Å resolution and belonged to the orthorhombic space group I222 or I212121, with unit-cell parameters a = 48.6, b = 61.9, c = 79.6 Å. The complete sequence and structure determination of amaryllin are in progress. © 2009 International Union of Crystallography. All rights reserved.

Acta Crystallographica Section F: Structural Biology and Crystallization Communications

Isolation, purification, crystallization and preliminary crystallographic studies of amaryllin, a plant pathogenesis-related protein from Amaryllis belladonna

International Research Journal of Social Sciences, Puducherry

Adolescence and Sexual Risk Behaviour

The peptide Boc-Ile-ΔAla-OCH3 was synthesized and its three dimensional structure was determined using crystals grown under two different conditions using solvents (a) ethyl acetate and (b) an 80:20 mixture of dichloromethane and heptane. In the first case, the peptide crystallized in space group P21 with six crystallographically independent molecules while in the second condition the peptide crystallized in space group P32 with four molecules in the asymmetric unit. In both structures the conformations of all the crystallographically independent molecules were almost identical. ΔAla adopted a planar conformation with the φ torsion angle having values centred at ±180°in all the molecules. The crystal structures in two different crystalline states showed that ΔAla induced a distorted inverse γ-turn in the preceding residue with φ and ψ torsions centred at 100(1) and -125(1)°, respectively. The molecular packing in both crystal forms generated the sheet-like structures that were stabilized by intermolecular hydrogen bonds between the layers of molecules. © 2004 Elsevier B.V. All rights reserved.

Journal of Molecular Structure

Design rules for peptides with α, β-dehydro-residues: Synthesis of a model peptide Boc-Ile-ΔAla-OCH3 and its crystal structures obtained from two different solvents

The extensive efforts to screen thermophilic fungi and bacteria, isolated from various environmental samples, have resulted in the selection of Thermomucor indicae-seudaticae, Geobacillus thermaleovorons NP33 and G. thermoleovorans NP54 for the production of glucoamylase, amylopullulanase and α-amylase, respectively. Submerged and solid-state fermentation processes were optimized for maximizing the secretion of glucoamylase by T. indicae-seudaticoe. The production of amylopullulanase and α-amylase by NP33 and NP54 in submerged fermentation was also optimized. Glucoamylase was optimally active at pH 7.0 and 100°C and was shown to saccharify soluble as well as raw starches. Amylopullulanase and α-amylase exhibited optima at pH 7.0 and 100°C and saccharified starch efficiently. Differential inhibition and action on mixed substrates clearly suggested that there are two separate active sites for α-amylase and pullulanase activities of amylopullulanase. Both α-amylase and amylopullulanase are high maltose-forming and Ca2+-independent. These amylolytic enzymes have been shown to be useful in starch saccharification alone and in combination.

Biochemical Society Transactions

Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles

Indian Veterinary Journal

Fungal infection of horn in bullocks

The concentration of melanin determines the intensity of colors of the skin and hair of animals. Melanin pigments are tyrosine-based polymers formed in melanocytes within specialized organelles called melanosomes. In order to understand the mechanism of melanin polymerization, lactoferrin, a basic protein with a pI value of 9.0, has been used to produce melanin. Lactoferrin is a monomeric iron-binding protein with a molecular weight of 80 kDa. The crystals of lactoferrin were soaked in a solution containing dihydroxyphenylalanine (DOPA) and tyrosinase enzyme. These crystals were used for X-ray intensity data collection. The intensity data were collected to 2.7-Å resolution to an overall completeness of 91% with an Rsym of 0.071. The crystals belong to orthorhombic space group P212121 with cell dimensions: a = 85.0 Å, b = 99.8 Å, c = 103.4 Å. The structure was determined by molecular replacement method, using the model of diferric mare lactoferrin, and refined to an R-factor 0.215 (Rfree = 0.287) for all the data to 2.7-Å resolution. The final model comprises 5,281 protein atoms from 689 amino acids, 2Fe3+, 2CO32- ions, 2 indole-5,6-quinone molecules (IQ), and 73 water molecules. Two IQ molecules, one in each lobe, bind to lactoferrin. In the C-lobe, the IQ binds in the iron-binding cleft, whereas in the N-lobe, it is located in the side pocket between two α-helices, filled with solvent molecules in the native iron-saturated mare lactoferrin. The IQ molecules interact with protein molecule mainly through glutamic acid in both lobes, without significant perturbation to the protein structure. The orientation of N- and C-lobes in the present structure is similar to that observed in the native iron-saturated protein. However, as a result of the binding of IQ molecules, the orientations of the domains N1, N2 and C1, C2 in the two cases differ slightly. © 2001 Wiley-Liss, Inc.

Proteins: Structure, Function and Genetics

Lactoferrin-melanin interaction and its possible implications in melanin polymerization: Crystal structure of the complex formed between mare lactoferrin and melanin monomers at 2.7-Å resolution