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FEBS J. 2019 Jul 10. doi: 10.1111/febs.14993. [Epub ahead of print]

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

Author information

1
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad de México, 04510, Mexico.
2
Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Via G. Amendola 122/O, 70126, Bari, Italy.

Abstract

Chitinases are plant 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 E. 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 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. This article is protected by copyright. All rights reserved.

KEYWORDS:

Agave tequilana ; SAXS models; antifungal proteins; binding energetics; class I chitinases

PMID:
31291689
DOI:
10.1111/febs.14993

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