Araújo Castro Jacqueline, Gomes Ferreira Monique Drielle, Santana Silva Raner José, Andrade Bruno Silva, Micheli Fabienne
Universidade Estadual de Santa Cruz (UESC), Centro de Biotecnologia e Genética (CBG), Ilhéus, Bahia, Brazil.
Instituto Federal de Educação, Ciência e Tecnologia Baiano (IFBaiano), Santa Inês, Bahia, Brazil.
PLoS One. 2017 May 1;12(5):e0176878. doi: 10.1371/journal.pone.0176878. eCollection 2017.
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.
交替氧化酶（AOX）蛋白存在于植物、真菌、原生动物和一些无脊椎动物中。它参与线粒体呼吸链，为电子传递提供了一条替代途径，导致氧气还原形成水。本研究旨在从核苷酸和蛋白质水平对柑橘（克莱门氏小柑橘）和甜橙（脐橙）中的AOX基因家族进行表征，包括启动子分析、系统发育分析和脐橙基因表达分析。本研究还旨在对一种AOX异构体（CcAOXd）进行同源建模。此外，还进行了CcAOXd蛋白与泛醌（UQ）的分子对接。在每个柑橘品种中鉴定出四个AOX基因。这些基因的开放阅读框（ORF）长度在852 bp至1150 bp之间，外显子数量在4至9个之间。每个AOX基因上游1500 bp的区域包含与内部和外部响应因子相关的调控顺式元件。脐橙AOX基因在柑橘组织中表现出差异表达。所有AOX蛋白预计都位于线粒体中。它们包含保守基序LET、NERMHL、LEEEA和RADE-H以及几个推定的翻译后修饰位点。通过与布氏锥虫的AOX同源建模得到CcAOXd蛋白（同一性为45%）。CcAOXd的三维结构显示存在两个疏水螺旋，可能参与该蛋白在线粒体内膜中的锚定。该蛋白的活性位点位于AOX结构内部深处的疏水环境中，包含一个双铁中心。CcAOXd与UQ的分子对接显示，结合位点是由螺旋形成并嵌入膜中的凹陷口袋。这些数据对于柑橘AOX基因和/或蛋白未来的功能研究以及利用UQ同源物进行AOX抑制的生物技术方法具有重要意义。
