A biophysical and structural study of two chitinases from Agave tequilana and their potential role as defense proteins

• Published in: The FEBS journal 2019-12, Vol.286 (23), p.4778-4796
• Main Authors: Sierra‐Gómez, Yusvel, Rodríguez‐Hernández, Annia, Cano‐Sánchez, Patricia, Gómez‐Velasco, Homero, Hernández‐Santoyo, Alejandra, Siliqi, Dritan, Rodríguez‐Romero, Adela
• Format: Article
• Language: English
• Subjects: Agave tequilana; Agave tequilana; Amino acids; Antifungal activity; antifungal proteins; binding energetics; Binding sites; Calorimetry; Carbohydrates; Chitin; Chitinase; class I chitinases; Colloid chitin; Computer simulation; Coupling (molecular); Domains; E coli; Enthalpy; Enzymes; Fungi; Fungicides; Fusarium oxysporum; Gene expression; Glycosidases; Glycoside hydrolase; Grooves; Homology; Hydrolase; Plant protection; Ribonucleic acid; RNA; SAXS models; Small angle X ray scattering; Titration; Titration calorimetry
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.14993

Plant chitinases are enzymes that have several functions, including providing protection against pathogens. Agave tequilana is an economically important plant that is poorly studied. Here, we identified a chitinase from short reads of the A. tequilana transcriptome (AtChi1). A second chitinase, differing by only six residues from the first, was isolated from total RNA of plants infected with Fusarium oxysporum (AtChi2). Both enzymes were overexpressed in Escherichia coli and analysis of their sequences indicated that they belong to the class I glycoside hydrolase family19, whose members exhibit two domains: a carbohydrate‐binding module and a catalytic domain, connected by a flexible linker. Activity assays and thermal shift experiments demonstrated that the recombinant Agave enzymes are highly thermostable acidic endochitinases with Tm values of 75 °C and 71 °C. Both exhibit a molecular mass close to 32 kDa, as determined by MALDI‐TOF, and experimental pIs of 3.7 and 3.9. Coupling small‐angle x‐ray scattering information with homology modeling and docking simulations allowed us to structurally characterize both chitinases, which notably show different interactions in the binding groove. Even when the six different amino acids are all exposed to solvent in the loops located near the linker and opposite to the binding site, they confer distinct kinetic parameters against colloidal chitin and similar affinity for (GlnNAc)6, as shown by isothermal titration calorimetry. Interestingly, binding is more enthalpy‐driven for AtChi2. Whereas the physiological role of these chitinases remains unknown, we demonstrate that they exhibit important antifungal activity against chitin‐rich fungi such as Aspergillus sp. Database SAXS structural data are available in the SASBDB database with accession numbers SASDDE7 and SASDDA6. Enzymes Chitinases (EC3.2.1.14). AtChi1 and AtChi2 are thermostable class 1 chitinases that are catalytically active against colloidal chitin and are involved in defense mechanisms of Agave tequilana. Despite exhibiting only six different residues in their sequences, they showed variations in their antifungal activity, as well as catalytic and binding behavior against chito‐oligosaccharides. SAXS analysis showed the spatial arrangement of the two domains present in these enzymes.