High-resolution structure of a papaya plant-defense barwin-like protein solved by in-house sulfur-SAD phasing.

The first crystal structure of a barwin-like protein, named carwin, has been determined at high resolution by single-wavelength anomalous diffraction (SAD) phasing using the six intrinsic S atoms present in the protein. The barwin-like protein was purified from Carica papaya latex and crystallized in the orthorhombic space group P212121. Using in-house Cu Kα X-ray radiation, 16 cumulative diffraction data sets were acquired to increase the signal-to-noise level and thereby the anomalous scattering signal. A sequence-database search on the papaya genome identified two carwin isoforms of 122 residues in length, both containing six S atoms that yield an estimated Bijvoet ratio of 0.93% at 1.54 Šwavelength. A systematic analysis of data quality and redundancy was performed to assess the capacity to locate the S atoms and to phase the data. It was observed that the crystal decay was low during data collection and that successful S-SAD phasing could be obtained with a relatively low data multiplicity of about 7. Using a synchrotron source, high-resolution data (1 Å) were collected from two different crystal forms of the papaya latex carwin. The refined structures showed a central ß-barrel of six strands surrounded by several α-helices and loops. The ß-barrel of carwin appears to be a common structural module that is shared within several other unrelated proteins. Finally, the possible biological function of the protein is discussed.

Ceramide, cerebroside and triterpenoid saponin from the bark of aerial roots of Ficus elastica (Moraceae).

Three compounds, ficusamide (1), ficusoside (2) and elasticoside (3), were isolated from the bark of aerial roots of Ficus elastica (Moraceae), together with nine known compounds, including four triterpenes, three steroids and two aliphatic linear alcohols. The chemical structures of the three compounds were established by extensive 1D and 2D NMR spectroscopy, mass spectrometry and by comparison with published data. The growth inhibitory effect of the crude extract and isolated compounds was evaluated against several microorganisms and fungi. The cytotoxicity against human cancer cell lines was also assessed. Ficusamide (1) displayed a moderate in vitro growth inhibitory activity against the human A549 lung cancer cell line and a strong activity against Staphylococcus saprophyticus, while elasticoside (3) showed a potent activity on Enterococcus faecalis.

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs.

BACKGROUND: Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily. RESULTS: Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a ß-hairpin pointed towards the substrate binding cleft. The invariant ß-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism. CONCLUSIONS: The seven families and subfamilies of the lysozyme superfamily all have in common a ß-hairpin structure which displays a family-specific sequence motif. These GH ß-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.

Purification and characterization of a wound-inducible thaumatin-like protein from the latex of Carica papaya.

A 22.137 kDa protein constituent of fresh latex was isolated both from the latex of regularly damaged papaya trees and from a commercially available papain preparation. The protein was purified up to apparent homogeneity and was shown to be absent in the latex of papaya trees that had never been previously mechanically injured. This suggests that the protein belongs to pathogenesis-related protein family, as expected for several other protein constituents of papaya latex. The protein was identified as a thaumatin-like protein (class 5 of the pathogenesis-related proteins) on the basis of its partial amino acid sequence. By sequence analysis of the Carica genome, three different forms of thaumatin-like protein were identified, where the latex constituent belongs to a well-known form, allowing the molecular modeling of its spatial structure. The papaya latex thaumatin-like protein was further characterized. The protein appears to be stable in the pH interval from 2 to 10 and resistant to chemical denaturation by guanidium chloride, with a DeltaG(water)(0) of 15.2 kcal/mol and to proteolysis by the four papaya cysteine proteinases. The physiological role of this protein is discussed.

X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases.

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.

Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex.

A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 A, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-D-glucosamine, the monomer resulting from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P2(1), with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 A, beta = 95.33 degrees and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 A. Structure refinement is currently in progress.

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the alpha-chymotrypsin-proflavin interaction.

Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K(obs), DeltaH(obs)(0) ). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between alpha-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between alpha-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria.

Affinity chromatography: a useful tool in proteomics studies.

Separation or fractionation of a biological sample in order to reduce its complexity is often a prerequisite to qualitative or quantitative proteomic approaches. Affinity chromatography is an efficient protein separation method based on the interaction between target proteins and specific immobilized ligands. The large range of available ligands allows to separate a complex biological extract in different protein classes or to isolate the low abundance species such as post-translationally modified proteins. This method plays an essential role in the isolation of protein complexes and in the identification of protein-protein interaction networks. Affinity chromatography is also required for quantification of protein expression by using isotope-coded affinity tags.

Structural characterization of the papaya cysteine proteinases at low pH.

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.

Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases.

Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7A resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed beta-propeller, with two additional alpha-helices and one beta hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded beta-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the beta-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase.

Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex.

In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 A and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 A.

Detection of three wound-induced proteins in papaya latex.

The effects of routine mechanical wounding for latex collection from unripe fruits of the tropical Carica papaya tree were investigated. For that purpose, the protein composition of three different latexes was analyzed. The first one, commercially available, was provided in the form of a spray-dried powder, the second one was harvested from fully grown but unripe papaya fruits that are regularly tapped for latex production and the last one, was obtained from similar fruits wounded for the first time. Repeated mechanical wounding was found to profoundly affect the protein content of the latex inducing, among others, activation of papain. Regularly tapped latexes also accumulated several low molecular weight proteins not yet identified, as well as three proteins identified as a trypsin inhibitor, a class-II chitinase and a glutaminyl cyclase on the basis of their enzymatic or inhibitory activities and chromatographic elution profiles. This latter was found here, for the first time, to be a wound-induced protein. The roles of these proteins in the plant defense mechanism are discussed.

Fractionation and purification of the enzymes stored in the latex of Carica papaya.

The latex of the tropical species Carica papaya is well known for being a rich source of the four cysteine endopeptidases papain, chymopapain, glycyl endopeptidase and caricain. Altogether, these enzymes are present in the laticifers at a concentration higher than 1 mM. The proteinases are synthesized as inactive precursors that convert into mature enzymes within 2 min after wounding the plant when the latex is abruptly expelled. Papaya latex also contains other enzymes as minor constituents. Several of these enzymes namely a class-II and a class-III chitinase, an inhibitor of serine proteinases and a glutaminyl cyclotransferase have already been purified up to apparent homogeneity and characterized. The presence of a beta-1,3-glucanase and of a cystatin is also suspected but they have not yet been isolated. Purification of these papaya enzymes calls on the use of ion-exchange supports (such as SP-Sepharose Fast Flow) and hydrophobic supports [such as Fractogel TSK Butyl 650(M), Fractogel EMD Propyl 650(S) or Thiophilic gels]. The use of covalent or affinity gels is recommended to provide preparations of cysteine endopeptidases with a high free thiol content (ideally 1 mol of essential free thiol function per mol of enzyme). The selective grafting of activated methoxypoly(ethylene glycol) chains (with M(r) of 5000) on the free thiol functions of the proteinases provides an interesting alternative to the use of covalent and affinity chromatographies especially in the case of enzymes such as chymopapain that contains, in its native state, two thiol functions.

Evidence that thermodynamic stability of papaya glutamine cyclase is only marginal.

Papaya glutamine cyclase (PQC), a glycoprotein with a molecular mass of 32,980 Da, is a minor constituent of the papaya latex protein fraction. In neutral aqueous solutions, PQC adopts an all-beta conformation and exhibits high resistance to both proteolysis and denaturation. Complete unfolding of PQC requires a combination of an acidic medium and chemical denaturant such as urea or guanidine hydrochloride. The unfolding process takes place through formation of an intermediate A state that accumulates in the absence of chemical denaturants and displays all the features of a molten globule state. The different conformational states-N (native), A (acid-inactivated), and U (unfolded)-have been characterized by means of circular dichroism measurements, fluorescence spectroscopies, Stokes radii determinations, and 8-anilino-1-naphtalenesulfonic acid (ANS) binding characteristics. The unfolding pathways of the enzyme was further studied to estimate thermodynamic parameters characterizing both transitions N if A and A if U. In its A state, PQC is catalytically inefficient and highly susceptible to proteolysis. Also, its thermodynamic stability is decreased by some 3-5 kcal/mol. Conversion of the native to the A state involves digging up of five amino functions together with protonation of four to five acidic groups with pK(a)s, in the native state, around 2.7. It proceeds both cooperatively and reversibly although, in vitro, the refolding process is slow. Unfolding of the A state, on the other hand, occurs with a low degree of cooperativity. The intermediate A state thus seems to be only marginally more stable than the unfolded state. The role of suspected internal ion pairs in the stabilization of the native state of this enzyme is discussed.