Alternative oxidase (AOX) constitutes a small family of proteins in Citrus clementina and Citrus sinensis L. Osb

Abstract

The alternative oxidase (AOX) protein is present in plants, fungi, protozoa and some invertebrates. It is involved in the mitochondrial respiratory chain, providing an alternative route for the transport of electrons, leading to the reduction of oxygen to form water. The present study aimed to characterize the family of AOX genes in mandarin (Citrus clementina) and sweet orange (Citrus sinensis) at nucleotide and protein levels, including promoter analysis, phylogenetic analysis and C. sinensis gene expression. This study also aimed to do the homology modeling of one AOX isoform (CcAOXd). Moreover, the molecular docking of the CcAOXd protein with the ubiquinone (UQ) was performed. Four AOX genes were identified in each citrus species. These genes have an open reading frame (ORF) ranging from 852 bp to 1150 bp and a number of exons ranging from 4 to 9. The 1500 bp-upstream region of each AOX gene contained regulatory cis-elements related to internal and external response factors. CsAOX genes showed a differential expression in citrus tissues. All AOX proteins were predicted to be located in mitochondria. They contained the conserved motifs LET, NERMHL, LEEEA and RADE-H as well as several putative post-translational modification sites. The CcAOXd protein was modeled by homology to the AOX of Trypanosona brucei (45% of identity). The 3-D structure of CcAOXd showed the presence of two hydrophobic helices that could be involved in the anchoring of the protein in the inner mitochondrial membrane. The active site of the protein is located in a hydrophobic environment deep inside the AOX structure and contains a diiron center. The molecular docking of CcAOXd with UQ showed that the binding site is a recessed pocket formed by the helices and submerged in the membrane. These data are important for future functional studies of citrus AOX genes and/or proteins, as well as for biotechnological approaches leading to AOX inhibition using UQ homologs.

Caracterização Da Enzima Oxidase Alternativa Do Fungo Moniliophthora Perniciosa Com O Auxilio Das Ferramentas De Bioinformática / Characterization of the Enzyme Oxidase Alternative of the Fungus Moniliophthora Perniciosa With the Aid of Bioinformatics Tools

“…Other similar works, such as that of Castro et al, 2017 also characterized the three-dimensional AOX structure of some citrus plants (C. Clementina, C. Sinensis), and observed the conservation of some AOX domains involved in the mitochondrial respiratory chain providing a alternative route for electron transport (via AOX alternative) [21].…”

Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors

“…These techniques have also enabled study of the domains involved in enzymatic catalysis and of the topography of the mitochondrial membrane enzyme and its post-translational regulation, as well as reconstruction of the evolution of the enzyme in different taxonomic groups 21 . The genes encoding AOX have been found in all higher plants and in some algae, fungi, protists and invertebrates, but not in vertebrates 14 , 15 , 22 , 66 , 67 . We have found evidence of the existence of an alternative oxidase pathway in the mitochondria of the fish parasite P. dicentrarchi , by using functional physiological techniques, which measure oxygen consumption after the addition of inhibitors of the cytochrome pathway, and immunochemical assays, which use polyclonal antibodies against a fully conserved C-terminal consensus motif from isoforms 1 and 2 of plant AOX 13 .…”

“…The typical structure of the AOX gene is characterized by the presence of four exons separated by three gene-specific introns of different lengths 70 , 71 . After transcription and splicing, the AOX gene in P. dicentrarchi generated a mature mRNA that encodes a protein of an estimated molecular weight of 35,638.68 daltons, a size like the standard sizes of the AOXs of various organisms ranging between 32–36 kDa 15 . AOX is an integral interfacial membrane protein that interacts with a single leaflet of the lipid bilayer 26 .…”

“…In plants, AOX is considered a ubiquitous enzyme; however, in algae 3 , fungi 4 – 9 , protozoa 10 – 13 and several invertebrate species 14 it is quite sporadic. AOX is involved in the mitochondrial respiratory chain and provides an alternative pathway for the direct transfer of electrons from ubiquinone to oxygen, thus forming water 15 . It facilitates mitochondrial respiratory activity and therefore the functioning of the Krebs cycle after inhibition of the cytochrome route due to the presence of nitric oxide, cyanide (both inhibitors of the complex IV) or antimycin A (complex III inhibitor) 1 , 12 , 16 .…”

Employing CRISPR/Cas Technology for the Improvement of Potato and Other Tuber Crops

“…At the beginning of the 20th century, the AOX was first discovered from thermogenic plants during anthesis ( Saha et al, 2016 ). Later it was found that it exists in the inner mitochondrial membrane of plants, fungi, protozoa, and some invertebrates ( Araújo Castro et al, 2017 ). AOX activity can protect cells from reactive oxygen species (ROS) such as O 2 – , H 2 O 2 , and OH, particularly during different abiotic stress ( Wang and Vanlerberghe, 2013 ).…”