title: Tc-cAPX, a cytosolic ascorbate peroxidase of Theobroma cacao L. engaged in the interaction with Moniliophthora perniciosa, the causing agent of witches' broom disease.
abstract: The level of hydrogen peroxide (H2O2) in plants signalizes the induction of several genes, including that of ascorbate peroxidase (APX-EC 1.11.1.11). APX isoenzymes play a central role in the elimination of intracellular H2O2 and contribute to plant responses to diverse stresses. During the infection process in Theobroma cacao by Moniliophthora perniciosa oxidative stress is generated and the APX action recruited from the plant. The present work aimed to characterize the T. cacao APX involved in the molecular interaction of T. cacao-M. perniciosa. The peroxidase activity was analyzed in protein extracts from cocoa plants infected by M. perniciosa and showed the induction of peroxidases like APX in resistant cocoa plants. The cytosolic protein of T. cacao (GenBank: ABR68691.2) was phylogenetically analyzed in relation to other peroxidases from the cocoa genome and eight genes encoding APX proteins with conserved domains were also analyzed. The cDNA from cytosolic APX was cloned in pET28a and the recombinant protein expressed and purified (rTc-cAPX). The secondary structure of the protein was analyzed by Circular Dichroism (CD) displaying high proportion of α-helices when folded. The enzymatic assay shows stable activity using ascorbate and guaiacol as an electron donor for H2O2 reduction. The pH 7.5 is the optimum for enzyme activity. Chromatographic analysis suggests that rTc-cAPX is a homodimer in solution. Results indicate that the rTc-cAPX is correctly folded, stable and biochemically active. The purified rTc-cAPX presented biotechnological potential and is adequate for future structural and functional studies.
authors.affiliation: Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz - UESC, Rodovia Jorge Amado Km 16, 45662-900 Ilhéus, BA, Brazil.
authors.forename: Luciana Rodrigues
authors.initials: LR
authors.lastname: Camillo
authors.forename: Ciro Ribeiro
authors.initials: CR
authors.lastname: Filadelfo
authors.forename: Paulo Sérgio
authors.initials: PS
authors.lastname: Monzani
authors.forename: Ronan Xavier
authors.initials: RX
authors.lastname: Corrêa
authors.forename: Karina Peres
authors.initials: KP
authors.lastname: Gramacho
authors.forename: Fabienne
authors.initials: F
authors.lastname: Micheli
authors.forename: Carlos Priminho
authors.initials: CP
authors.lastname: Pirovani
mesh_terms: D000363:Agaricales; D060387:Ascorbate Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002099:Cacao / Q000201:enzymology / Q000235:genetics / Q000382:microbiology; D003600:Cytosol; D018076:DNA, Complementary; D019281:Dimerization; D060467:Disease Resistance / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018384:Oxidative Stress; D010802:Phylogeny; D010935:Plant Diseases / Q000382:microbiology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D060387:Ascorbate Peroxidases
keywords: Ascorbate peroxidase-related (APX-R); Enzyme activity; Enzyme characterization; Hydrogen peroxide; Oxidative stress; Peroxidase; Recombinant protein