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Fig. 6 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 6. Morphological alterations induced in Lasiodiplodia theobromae hyphae by Anacardium occidentale chitinase. SEM images of untreated mycelium(A-B), mycelium treated with 500 μg AoChi (C–I) and mycelium treated with 5 μg Carbendazim (J–M) are shown. The assays to evaluate the morphological alterations in L. theobromae grown in vitro in the presence of AoChi and acquisition of SEM images were performed as described in the Methods section. SEM images from fungus grown in the presence of water (A–B) or Carbendazim (J–M) were included for comparison.

Fig. 7 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 7. Three-dimensional molecular model of AoChi and its interaction with a chitin oligomer. (A) Cartoon representation of the three-dimensional molecular model of AoChi, which was generated by homology modeling. Side chains of putative catalytic residues are shown as sticks. Disulfide bonds are colored orange. (B) Detailed view of a chito-oligosaccharide (yellow) docked in the substrate-binding cleft of AoChi. Side chains of AoChi residues that probably interact with the docked ligand through hydrogen bonds (represented as yellow dotted lines) are shown as sticks (cyan). N and O atoms are colored blue and red, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 5. Effects of temperature (A), pH (B), metal ions (C) and chemical reagents (D) on the hydrolytic activity (A–D) and stability (A–B) of AoChi. Enzymatic assays were performed using the purified recombinant protein (200 μg/mL) and colloidal chitin as substrate, as described in the Methods section. In panels C and D, means that are significantly different (P <0.05; Bonferroni's multiple comparisons test) when compared to control are indicated by asterisks.

Fig. 4 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 4. Polyacrylamide gel electrophoresis (SDS-PAGE) of the purified recombinant chitinase from Anacardium occidentale, a member of class VI that has chitinolytic activity. (A) The recombinant chitinase (lane 1; 35 μg) was reduced with β-mercaptoethanol and subjected to denaturing gel electrophoresis (15% polyacrylamide), as described in the Methods section. Lane M: protein markers. (B) Representative fragmentation mass spectrum (LC-MS/MS) of a tryptic peptide, obtained from in gel-digestion of the 45 kDa protein band, that matched a segment of 17 residues of the primary structure of the recombinant product. (C) Colorimetric enzymatic assay, showing the ability of the recombinant cashew chitinase to degrade colloidal chitin. (D) Plot of initial reaction velocities vs substrate concentration. The enzymatic assays (panels C and D) were performed at pH 6.0 and 40 ◦C, and using colloidal chitin as substrate, as described in the Methods section.

Fig. 3 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 3. Alignment of the amino acid sequence of the CatD of AoChi with known three-dimensional structures of GH19 chitinases from plants. The CatD sequence of AoChi was aligned to GH19 structures from: Carica papaya (PDB ID: 3CQL), Vigna unguiculata subsp. sesquipedalis (PDB ID: 4TX7), Secale cereale (PDB ID: 4DWX), Hordeum vulgare (PDB ID: 2BAA), Oryza sativa subsp. japonica (PDB ID: 2DKV), Canavalia ensiformis (PDB ID: 1DXJ), Brassica juncea (PDB ID: 2Z37), Cryptomeria japonica (PDB ID: 5H7T) and Picea abies (PDB ID: 3HBD). Sites containing residues involved in catalysis (triangles) and substrate-binding (hash marks) are indicated. Disulfide bonds, as observed in the three-dimensional model of AoChi (Fig. 7), are indicated by orange lines. Loop regions (I–V), as assigned by Taira et al. (Taira et al., 2011), are boxed. Loop VI (this work), which is present only in AoChi and other class VI chitinases, is also boxed. Alignment columns are colored according to the ALSCRIPT Calcons convention, as implemented in ALINE (Bond and Schüttelkopf, 2009), using a predefined color scheme, which reflects the conservation of amino acid properties in each column (dark blue: identical residues; white: dissimilar residues). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 2. Maximum likelihood (ML) tree showing the phylogenetic relationship of Anacardium occidentale chitinase with representative plant chitinases from classes I, II, IV, VI and VII. The evolutionary history of the sequences was inferred by using the ML method and Whelan and Goldman + Freq. model (Whelan and Goldman, 2001). The tree with the highest log likelihood (12837.58) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.5328)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 3.91% sites). The percentage of trees in which the associated sequences clustered together, as determined by the bootstrap test (100 replicates), is shown next to the branches. This analysis involved 62 amino acid sequences, and the final dataset contained a total of 371 positions (Table S2 and Fig. S7). Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

Fig. 6 in TIM barrel fold and glycan moieties in the structure of ICChI, a protein with chitinase and lysozyme activity

Fig. 6. (A) Three-dimensional (3D) structural model of the ICChI- NAG complex illustrating by Docking. (B) DIMPLOT result revels the interacting amino acid residue during formation of complex.

Fig. 1 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 1. Domain architecture of Anacardium occidentale chitinase (AoChi). (A) Graphical representation of AoChi domains (SP: signal peptide; GH19 CatD: GH19 catalytic domain). The catalytic residues of AoChi, Lys(K)128, Glu(E)150 and Tyr(Y)183, are highlighted. (B) Alignment of a stretch of 61 residues from the primary structure of AoChi (from His124 to Trp184), which contains the 3 catalytic residues, with corresponding segments from homologous proteins belonging to plant species from different families and orders (Table S5). Alignment columns are colored according to the ALSCRIPT Calcons convention, as implemented in ALINE (Bond and Schüttelkopf, 2009), using a predefined color scheme, which reflects the conservation of amino acid properties in each column (dark red: identical residues; white: dissimilar residues). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in TIM barrel fold and glycan moieties in the structure of ICChI, a protein with chitinase and lysozyme activity

Fig. 2. The three dimensional crystal structure of ICChI where outer ball and stick (green-red) are glycan ligands, Alpha helices (red coil); parallel beta sheets (yellow arrow); random coils or loop (green); (A) topview of ICChI structure, (B) side view of ICChI structure.

Fig. 1 in TIM barrel fold and glycan moieties in the structure of ICChI, a protein with chitinase and lysozyme activity

Fig. 1. (A) Silver stained 12.5% SDS-PAGE gel of purified ICChI. Lane 1 contains molecular weight markers (Pageruler prestained protein ladder, Fermentas SM0671) and lane 2 represents pure and homogeneous ICChI protein shown by arrow. (B) Crystals of ICChI grown in 4–5 days in a hanging drop at 291 K equilibrated against 750 μl reservoir solution containing 0.005 M Cobalt chloride, 0.005 M Cadmium chloride, 0.005 M Magnesium chloride, 0.005 M Nickel chloride and 11% (w/v) PEG 3350 in 0.1 M HEPES buffer, pH 7.0. The tetragonal bipyramide-shaped crystals had a typical size of 300 × 200 × 200 μm. (C) X-ray diffraction from the crystal of ICChI protein produced interference pattern.

Fig. 5. The electron density and N in TIM barrel fold and glycan moieties in the structure of ICChI, a protein with chitinase and lysozyme activity

Fig. 5. The electron density and N-linked glycosylation sites. (A) Asparagine residue 45. (B) Asparagine residue 172. (C) Asparagine residue 194.

Fig. 4 in TIM barrel fold and glycan moieties in the structure of ICChI, a protein with chitinase and lysozyme activity

Fig. 4. The catalytic residues of the ICChI structure are: aspartate 125; glutamate 127 and tyrosine 184. The grey mesh is electron density whereas the amino acid residues are green.