Vellore Institute of Technology (VIT) is a private university located in&nbsp;Tamil Nadu, India. Founded in 1984, as Vellore Engineering College, the institution offers 20 undergraduate, 34 postgraduate, four integrated and four research programs. It has campuses in Vellore, Amravati, Bhopal and Chennai.

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Snake venoms are cocktails of enzymes and non-enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A 2. Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure-based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non-enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor-enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications. Snake venoms are cocktails of enzymes and non-enzymatic proteins. Here, we describe the structures of the common snake venom enzymes, namely, acetylcholinesterase, L-amino acid oxidase, phospholipase A2, serine proteinase, and metalloproteinase, and their complexes. We discuss the contribution of these structures in understanding the mechanisms of catalysis and inhibition as well as in the structure-based design of new, potent inhibitors © 2011 The Authors Journal compilation © 2011 FEBS.

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Centre for Bio-Separation and Technology

Vellore Campus

Department of Social Sciences

School of Social Sciences & Languages

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FEBS Journal

Enzymatic toxins from snake venom: Structural characterization and mechanism of catalysis

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.

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

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

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

A novel ligand-binding site with functional implications has been identified in phospholipase A2 (PLA2). The binding of non-steroidal anti-inflammatory agent indomethacin at this site blocks both catalytic and anti-coagulant actions of PLA2. A group IIA PLA2 has been isolated from Daboia russelli pulchella (Russell's viper) which is enzymatically active as well as induces a strong anti-coagulant action. The binding studies have shown that indomethacin reduces the effects of both anti-coagulant and pro-inflammatory actions of PLA2. A group IIA PLA2 was co-crystallized with indomethacin and the structure of the complex has been determined at 1.4 A° resolution. The structure determination has revealed the presence of an indomethacin molecule in the structure of PLA2 at a site which is distinct from the conventional substrate-binding site. One of the carboxylic group oxygen atoms of indomethacin interacts with Asp 49 and His 48 through the catalytically important water molecule OW 18 while the second carboxylic oxygen atom forms an ionic interaction with the side chain of Lys 69. It is well known that the residues, His 48 and Asp 49 are essential for catalysis while Lys 69 is a part of the anti-coagulant loop (residues, 54-77). Indomethacin binds in such a manner that it blocks the access to both, it works as a dual inhibitor for catalytic and anti-coagulant actions of PLA2. This new binding site in PLA2 has been observed for the first time and indomethacin is the first compound that has been shown to bind at this novel site resulting in the prevention of anti-coagulation and inflammation. Copyright {\textcopyright}2009 JohnWiley {\&} Sons, Ltd.

Journal of Molecular Recognition

Simultaneous inhibition of anti-coagulation and inflammation: Crystal structure of phospholipase A2 complexed with indomethacin at 1.4 A° resolution reveals the presence of the new common ligand-binding site

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