Sanjit Kumar

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

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 A2. 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.

Biochemical studies and crystal structure determination of dihydrodipicolinate synthase from Pseudomonas aeruginosa

The intracellular enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Pseudomonas aeruginosa is a potential drug target because it is essential for the growth of bacteria while it is absent in humans. Therefore, in order to design new compounds using structure based approach for inhibiting the function of DHDPS from P. aeruginosa (Ps), we have cloned, characterized biochemically and biophysically and have determined its three-dimensional structure. The gene encoding DHDPS (dapA) was cloned in a vector pET-28c(+) and the recombinant protein was overexpressed in the Escherichia coli host. The K(m) values of the recombinant enzyme estimated for the substrates, pyruvate and (S)-aspartate-β-semialdehyde [(S)-ASA] were found to be 0.90±0.13 mM and 0.17±0.02 mM, respectively. The circular dichroism studies showed that the enzyme adopts a characteristic β/α conformation which is retained up to 65°C. The fluorescence data indicated the presence of exposed tryptophan residues in the enzyme. The three-dimensional structure determination showed that DHDPS forms a homodimer which is stabilized by several hydrogen bonds and van der Waals forces at the interface. The active site formed with residues Thr44, Tyr107 and Tyr133 is found to be stereochemically suitable for catalytic function. It may be noted that Tyr107 of the catalytic triad belongs to the partner molecule in the dimer. The structure of the complex of PsDHDPS with (S)-lysine determined at 2.65 Å resolution revealed the positions of three lysine molecules bound to the protein.

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

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 (Russells 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 Å 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

Identification of a novel and potent inhibitor of phospholipase A(2) in a medicinal plant: crystal structure at 1.93A and Surface Plasmon Resonance analysis of phospholipase A(2) complexed with berberine

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93Å and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).

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

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 Å 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.

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.

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.

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

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 A resolution and belonged to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 48.6, b = 61.9, c = 79.6 A. The complete sequence and structure determination of amaryllin are in progress.

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

BackgroundPlants 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 α-amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of α-amylase GH13. A new form of XAIP (XAIP-II) that inhibits α-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.ResultsIn order to determine the structural basis of the enhanced potency of XAIP-II against α-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 Å resolution. The binding studies of XAIP-II with xylanase GH11 and α-amylase GH13 have been carried out with surface plasmon resonance (SPR).ConclusionThe 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 α3-β3 (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 α-amylase binding site, which consists of α-helices α6 (residues, 193 - 206), α7 (residues, 230 - 243) and loop β6-α6 (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 α-helix α6, Glu231 and His236 by Thr232 and Ser237 respectively in α-helix α7. As a result, XAIP-II binds to xylanase GH11 less favorably while it interacts more strongly with α-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 α-amylase GH13 respectively and those of 4.5 × 10-7 M and 3.6 × 10-6 M of XAIP with xylanase GH11 and α-amylase GH13 respectively.

FRONTAL MUCOCELE AS A CAUSE OF GROSS UNILATERAL PROPTOSIS.

Frontal mucocele is an uncommon cause of proptosis. We present one such case resulting in gross proptosis with marked bony erosion of roof of orbit and floor of the anterior cranial fossa. In the absence of CT scan facility and ENT surgical expertise, the surgical procedure entailing drainage of mucoele and obliteration of cavity using abdominal fat, has resulted in a very satisfying outcome. Case Report A 34 year Sierra Lconean national was referred to our level III facility with history of painless, slowly growing mass over the left forehead of 2 years duration. There was no history of antecedent trauma. On examination there was a 12cm×8cm tense swelling over the left frontal region. It was non tender, non reducible, there were no transmitted impulses and local temperature was not raised. There was associated gross proptosis with the globe pushed down and out. Visual acuity in left eye was reduced to 6/36. There was relative afferent pupillary defect, papilledema and macular edema. Ocular movements were restricted in all directions. Haematological parameters and urinalysis were within normal limits. Radiograph of paranasal sinuses revealed erosion of roof of orbit and increased size of sinus cavity. Ultrasonography revealed echogenic debris and eroded bone. Facilities for CT scan/MRI were not available within the country and the destitute patient could not travel abroad to get these investigations done. Needle aspiration resulted in a mucoid aspirate, which was culture negative. A provisional diagnosis of a frontal mucocele was made and the patient was subjected to surgery. A curvilinear incision was given 1 cm above and parallel to the eyebrow. All the mucoid material was aspirated and diseased mucosa, which could be removed, was removed. The entire orbital roof was eroded and the globe could be “pushed out” using the index finger. Postero-superiorly the brain pulsations could be seen through the dura. The cavity did not reveal any growth or vascularised tissue. A PTFE coated biocompatible tube (suction drain) with multiple fenestrations was passed through the inferomedial recess into the nose and anchored there. Through a left anterior abdominal wall incision, fat was removed and used to obliterate the sinus cavity. Post operatively the patient was kept on antibiotics and analgesics and discharged on the 5th day. Drainage tube was removed after 3 months. Visual acuity left eye had improved to 6/18 and papilloedema regressed. At the end of another 3 months follow up there had been no recurrence of the mucocele. Fig. 1 12 × 8 cm swelling over left frontal region Fig. 2 Post operative photograph, no recurrence at 3 months follow up Discussion Mucoceles were first described by Langenbeck (1820) under the name of hydatides, and Rollet (1909) suggested the name mucocele. Mucoceles have been described as collections of mucous enclosed in a sac of lining sinus epithelium within an air sinus resulting from an obstruction to the outlet of the cavity which may cause an expansion of the sinus by resorption of the bony walls [1]. The paired frontal sinuses are lined with pseudostratified ciliated columnar epithelium, which provides mucous secretion to trap bacteria. This secretion then extrudes through the ostium via the nasofrontal duct into the infundibulum to be swallowed or expectorated. Frontal sinus mucoceles develop secondary to a blockage of the nasofrontal duct which may be secondary to infection, allergy, trauma, tumours, congenitally narrowed ostium or previous sinus surgery [2]. In nearly 30% cases no prior nasal symptoms can be elicited. As the lesion expands, there is thinning and erosion of the walls of the frontal sinus. Primarily the orbital roof is eroded resulting in proptosis, diplopia, periorbital or upper lid swelling, decreased visual acuity and restricted ocular movements, causing the patient to first seek an ophthalmologists opinion, as it happened in our case [3]. Incidence of mucocele of the paranasal sinuses as a cause of unilateral proptosis has been estimated to be between 1.5%-4% [4], making it a rare etiological factor of proptosis. In perhaps the largest series of 98 patients of fronto-ethmoidal mucoceles covering a period between 1962-1986, Lund et al found proptosis in 91 % of patients (average 6 mm) and lateral and inferior displacement in only about half the patients [5], thus indicating the usual small size of these lesions. Our patient in comparison had a huge mucocele and, therefore is a rarer entity. In a series of 46 patients covering a 13 year period, Evans (1981) found preoperative evidence of bony erosion in 34 (74%) patients. The floor of the frontal sinus was involved in 31(91%) and only 13(38%) had loss of posterior wall with exposure of the dura [6]. The Howarth and Lynch surgical procedure emphasized the importance of removing all diseased mucosa and creating adequate drainage. However, it may not be possible to always remove all the mucosa as it happened in our case. Inferiorly there was the danger of prolapse of orbital fat and superiorly the dura would have been exposed. Failure to maintain long-term patency of the frontonasal recess is one of the chief causes of failure. This we achieved by using a PTFE coated biocompatible tube with multiple fenestrations which was left in situ for 3 months. It is also important to obliterate the sinus cavity. A variety of exogenous and endogenous materials have been used but abdominal fat has proved to be the most popular [7] and was used in our patient. Inspite of a massive proptosis vision was fairly well preserved probably because of the longer length of the optic nerve relative to the orbital axis allowing a degree of “stretch”, Fujitani et al (1984) state that visual loss associated with mucocele is rare, and once established the chances of recovery are slight. Our case showed an improvement of two lines on the Snellens chart probably as a result of resolution of papilloedema and macular edema following decompression of the orbit. Today the best surgical approach would probably be an endoscopic approach, considering the functional and cosmetic aspects. However, its drawbacks are the requirement of equipment, expertise and incomplete exposure of the sinus cavity.