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

Autor: Oliveira ST; Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil., Azevedo MIG; Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil., Cunha RMS; Centro de Ciências Agrárias e Biológicas, Universidade do Vale do Acaraú, Sobral, Ceará, Brazil., Silva CFB; Embrapa Agroindústria Tropical, Fortaleza, Ceará, Brazil., Muniz CR; Embrapa Agroindústria Tropical, Fortaleza, Ceará, Brazil., Monteiro-Júnior JE; Laboratório de Genética Molecular, Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil., Carneiro RF; Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil., Nagano CS; Departamento de Engenharia de Pesca, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil., Girão MS; Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil., Freitas CDT; Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil., Grangeiro TB; Laboratório de Genética Molecular, Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil. Electronic address: thalles@ufc.br.
Jazyk: angličtina
Zdroj: Phytochemistry [Phytochemistry] 2020 Dec; Vol. 180, pp. 112527. Date of Electronic Publication: 2020 Sep 29.
DOI: 10.1016/j.phytochem.2020.112527
Abstrakt: A partial cDNA sequence from Anacardium occidentale CCP 76 was obtained, encoding a GH19 chitinase (AoChi) belonging to class VI. AoChi exhibits distinct structural features in relation to previously characterized plant GH19 chitinases from classes I, II, IV and VII. For example, a conserved Glu residue at the catalytic center of typical GH19 chitinases, which acts as the proton donor during catalysis, is replaced by a Lys residue in AoChi. To verify if AoChi is a genuine chitinase or is a chitinase-like protein that has lost its ability to degrade chitin and inhibit the growth of fungal pathogens, the recombinant protein was expressed in Pichia pastoris, purified and biochemically characterized. Purified AoChi (45 kDa apparent molecular mass) was able to degrade colloidal chitin, with optimum activity at pH 6.0 and at temperatures from 30 °C to 50 °C. AoChi activity was completely lost when the protein was heated at 70 °C for 1 h or incubated at pH values of 2.0 or 10.0. Several cation ions (Al 3+ , Cd 2+ , Ca 2+ , Pb 2+ , Cu 2+ , Fe 3+ , Mn 2+ , Rb + , Zn 2+ and Hg 2+ ), chelating (EDTA) and reducing agents (DTT, β-mercaptoethanol) and the denaturant SDS, drastically reduced AoChi enzymatic activity. AoChi chitinase activity fitted the classical Michaelis-Menten kinetics, although turnover number and catalytic efficiency were much lower in comparison to typical GH19 plant chitinases. Moreover, AoChi inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, causing several alterations in hyphae morphology. Molecular docking of a chito-oligosaccharide in the substrate-binding cleft of AoChi revealed that the Lys residue (theoretical pK a  = 6.01) that replaces the catalytic Glu could act as the proton donor during catalysis.
(Copyright © 2020 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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