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

• Published in: The FEBS journal 2014-10, Vol.281 (19), p.4535-4554
• Main Authors: Martínez‐Caballero, Siseth, Cano‐Sánchez, Patricia, Mares‐Mejía, Israel, Díaz‐Sánchez, Angel G., Macías‐Rubalcava, Martha L., Hermoso, Juan A., Rodríguez‐Romero, Adela
• Format: Article
• Language: English
• Subjects: Alternaria - drug effects; Amino Acid Sequence; Antifungal Agents - chemistry; Antifungal Agents - pharmacology; antifungal protein; Base Sequence; Catalytic Domain; Chitin - chemistry; chitin affinity; Chitinases - biosynthesis; Chitinases - chemistry; Chitinases - genetics; Chitinases - pharmacology; Cloning, Molecular; Conserved Sequence; Crystal structure; Crystallography, X-Ray; Enzyme Stability; Hevea - enzymology; Hydrolysis; Microbial Sensitivity Tests; Molecular Sequence Annotation; Molecular Sequence Data; Plant biology; plant chitinase‐like protein; Plant Leaves - enzymology; Plant Proteins - biosynthesis; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - pharmacology; Plant resistance; Protein Binding; Protein Structure, Secondary; Proteins; thermostability; X‐ray crystal structure
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12962

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 Å, 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. Database The 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 chitin‐binding domain (CBD1). Nucleotide sequence data are available in the GenBank database under the accession numbers KF648872 (chi‐L1) and KF648873 (chi‐L2). Hevea brasiliensis expresses chitinases and chitinase‐like proteins (HbCLP) belonging to families GH18 and GH19. Here, we characterized two HbCLPs, consisting of a catalytic and a chitin‐binding domain, which are highly thermostable, suggesting that during evolution from catalytically active proteins to CLPs they sacrificed activity for stability. These proteins showed antifungal