Purification and characterization of an insect α-amylase inhibitor/endochitinase from seeds of Job's Tears (Coix lachryma-jobi)

Ary, Maria B.; Richardson, Michael; Shewry, Peter R.

doi:10.1016/0167-4838(89)90007-1

Cited by 50 publications

(36 citation statements)

References 19 publications

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“…In general, plant endochitinases are proteins of 25-35 kDa molecular weight, which occur as monomers and have either high or low isoelectric points (for review see Boller, 1988). However, the bifunctional endochitinase and a-amylase inhibitor from Job's tear consists of a dimer of 26 kDa subunits (Ary et a/., 1989 Classification and structure of chitinases Three classes of plant chitinases have been proposed based on the primary structure (Shinshi etal., 1990): class I chitinases are enzymes with a N-terminal cysteine-rich domain of approximately 40 amino acids and a highly conserved main structure, separated by a variable hinge region (Figure 1). Class II chitinases lack the N-terminal cysteine-rich domain but have a high amino acid sequence identity to the main structure of class 1 chitinases.…”

Section: Enzyme Activities Substrates and Biochemical Propertiesmentioning

confidence: 99%

Plant chitinases

Collinge¹,

Kragh²,

Mikkelsen³

et al. 1993

The Plant Journal

791

528

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Section: Enzyme Activities Substrates and Biochemical Propertiesmentioning

confidence: 99%

Plant chitinases

Collinge¹,

Kragh²,

Mikkelsen³

et al. 1993

The Plant Journal

791

528

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“…For the full-length HbCLPs the ligand produces similar changes after binding, as observed in saturation curves which exhibit an enhanced fluorescence intensity at the k max of each protein (337.1 nm for HbCLP1 and 335.8 nm for HbCLP2) (Fig. 8A) the binding of (GlcNAc) 3 produced an increase in the fluorescence intensity in both cases and a slight blue shift in k max (Fig. 8A).…”

Section: Binding Experiments Between the Hbclps And Nacetyl-chitotriomentioning

confidence: 63%

“…Therefore they express several defense-related proteins that include chitinases and CLPs, and other proteins that could also be involved in different events such as cell division or flower development [30]. In any case, multiple isoforms of either chitinases or CLPs with different functions and characteristics can be expressed under these conditions [1][2][3].…”

Section: Resultsmentioning

confidence: 99%

“…The binding affinities of HbCLP1, HbCLP2 and their respective individual domains (CatDs and CBDs) for (GlcNAc) 3 were determined by fluorescence spectroscopy experiments. Both HbCLPs have one tryptophan residue (Trp23) located in the CBD and two tryptophan residues in the substrate-binding groove of the CatD, which are Trp122 and Trp171 in HbCLP1 (Fig.…”

Section: Binding Experiments Between the Hbclps And Nacetyl-chitotriomentioning

confidence: 99%

“…Chitinases (EC 3.2.1.14) and chitinase-like proteins (CLPs) are widely distributed among all kingdoms of life, and they participate in several physiological processes, including pathogenesis, nutrition, growth regulation and immunity, among others [1][2][3]. Chitinases catalyze the hydrolysis of b-1,4-linkages between N-acetylglucosamine (GlcNAc) residues in chitin, the second most abundant polysaccharide in nature, whereas CLPs lack enzymatic activity due to mutations in one or more of their amino acids required for catalysis but retain their potential to bind chitin oligosaccharides.…”

Section: Introductionmentioning

confidence: 99%

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Comparative study of two GH19 chitinase‐like proteins from Hevea brasiliensis, one exhibiting a novel carbohydrate‐binding domain

et al. 2014

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Plants express chitinase and chitinase-like proteins (CLPs) belonging to the glycosyl hydrolases of the GH18 and GH19 families, which exhibit varied functions. CLPs in the GH18 family have been structurally and functionally characterized; however, there are no structures available for any member of the GH19 family. In this study, two CLPs of the GH19 family from the rubber tree Hevea brasiliensis (HbCLP1 and HbCLP2) were cloned, expressed and characterized. HbCLP1 was identical to the allergen Hev b 11.0101 previously described by others, while HbCLP2 was a novel isoform exhibiting an unusual half chitin-binding domain before the catalytic domain. Sequence alignments showed that in the two proteins the catalytic residues Glu117 and Glu147 in HbCLP1 and HbCLP2, respectively, were mutated to Ala, accounting for the lack of activity. Nonetheless, both CLPs bound chitin and chitotriose (GlcNAc) 3 with high affinities, as evaluated with chitin-affinity chromatography and tryptophan fluorescence experiments. The chitin-binding domains also bound chitotriose with even higher affinities. The crystal structures of the HbCLP1-isolated domains were determined at high resolution. The analysis of the crystallographic models and docking experiments using (GlcNAc) 6 oligosaccharides provides evidence of the residues involved in sugar binding. Endochitinase activity was restored in both proteins by mutating residues A117E (HbCLP1) and A147E (HbCLP2); the distance between the catalytic proton donor and the catalytic nucleophile in the in silico mutated residues was 9.5 A, as occurs in inverting enzymes. HbCLP1 and HbCLP2 were highly thermostable and exhibited antifungal activity against Alternaria alternata, suggesting their participation in plant defense mechanisms. DatabaseThe atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4MST for the catalytic domain (CatD1) and 4MPI for the chitinbinding domain (CBD1). Nucleotide sequence data are available in the GenBank database under the accession numbers KF648872 (chi-L1) and KF648873 (chi-L2). AbbreviationsCatD1, catalytic domain of HbCLP1; CatD2, catalytic domain of HbCLP2; CatD, catalytic domain; CBD1, chitin-binding domain of HbCLP1; CBD2, chitin-binding domain of HbCLP2; CBD, chitin-binding domain; CLP, chitinase-like protein; GH, glycoside hydrolase; GlcNAc, Nacetylglucosamine; HbCLP, chitinase-like protein from Hevea brasiliensis; IPTG, isopropyl 1-thio-b-D-galactopyranoside; ITC, isothermal titration calorimetry; MES, 2-(N-morpholino)ethanesulfonic acid; NMR, nuclear magnetic resonance; PRP, pathogenesis-related proteins.

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1H NMR structure of an antifungal γ-thionin protein SIα1: Similarity to scorpion toxins

et al. 1998

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The three-dimensional structure of the Sorghum bicolor seed protein gamma-thionin SIalpha1 has been determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i+2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins.

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Purification and characterization of an insect α-amylase inhibitor/endochitinase from seeds of Job's Tears (Coix lachryma-jobi)

Cited by 50 publications

References 19 publications

Plant chitinases

Plant chitinases

Comparative study of two GH19 chitinase‐like proteins from Hevea brasiliensis, one exhibiting a novel carbohydrate‐binding domain

1H NMR structure of an antifungal γ-thionin protein SIα1: Similarity to scorpion toxins

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