Reinbothe Steffen, Bartsch Sandra, Rossig Claudia, Davis Manli Yang, Yuan Shu, Reinbothe Christiane, Gray John
Laboratoire de Génétique Moléculaire des Plantes and Biologie Environnementale et Systémique (BEeSy), Université Grenoble Alpes, Grenoble, France.
OraSure Technologies Inc., Bethlehem, PA, United States.
Front Plant Sci. 2019 May 15;10:593. doi: 10.3389/fpls.2019.00593. eCollection 2019.
Higher plants contain a small, 5-member family of Rieske non-heme oxygenases that comprise the inner plastid envelope protein TIC55, phaeophorbide oxygenasee (PAO), chlorophyllide oxygenase (CAO), choline monooxygenase, and a 52 kDa protein (PTC52) associated with the precursor NADPH:protochlorophyllide (Pchlide) oxidoreductase A (pPORA) A translocon (PTC). Some of these chloroplast proteins have documented roles in chlorophyll biosynthesis (CAO) and degradation (PAO and TIC55), whereas the function of PTC52 remains unresolved. Biochemical evidence provided here identifies PTC52 as Pchlide oxygenase of the inner plastid envelope linking Pchlide synthesis to pPORA import. Protochlorophyllide is the preferred substrate of PORA and its lack no longer allows pPORA import. The Pchlide -dependent import pathway of pPORA thus operates in etiolated seedlings and is switched off during greening. Using dexamethasone-induced RNA interference (RNAi) we tested if PTC52 is involved in controlling both, pPORA import and Pchlide homeostasis . As shown here, plants deprived of transcript and PTC52 protein were unable to import pPORA and died as a result of excess Pchlide accumulation causing singlet oxygen formation during greening. In genetic studies, no homozygous knock-out mutants could be obtained presumably as a result of embryo lethality, suggesting a role for PTC52 in the initial greening of plant embryos. Phylogenetic studies identified PTC52-like genes amongst unicellular photosynthetic bacteria and higher plants, suggesting that the biochemical function associated with PTC52 may have an ancient evolutionary origin. PTC52 also harbors conserved motifs with bacterial oxygenases such as the terminal oxygenase component of 3-ketosteroid 9-alpha-hydroxylase (KshA) from . 3D-modeling of PTC52 structure permitted the prediction of amino acid residues that contribute to the substrate specificity of this enzyme. -mutagenesis was used to test the predicted PTC52 model and provide insights into the reaction mechanism of this Rieske non-heme oxygenase.
高等植物含有一个由5个成员组成的小家族，即 Rieske 非血红素加氧酶，其中包括质体内膜蛋白TIC55、脱镁叶绿酸加氧酶（PAO）、叶绿素酸加氧酶（CAO）、胆碱单加氧酶，以及一种与前体NADPH：原叶绿素酸（Pchlide）氧化还原酶A（pPORA）转运体（PTC）相关的52 kDa蛋白（PTC52）。这些叶绿体蛋白中的一些在叶绿素生物合成（CAO）和降解（PAO和TIC55）中具有已被记录的作用，而PTC52的功能仍未明确。本文提供的生化证据表明，PTC52是质体内膜的Pchlide加氧酶，将Pchlide合成与pPORA导入联系起来。原叶绿素酸是PORA的首选底物，缺乏它就不再允许pPORA导入。因此，pPORA的依赖于Pchlide的导入途径在黄化幼苗中起作用，并在绿化过程中关闭。使用地塞米松诱导的RNA干扰（RNAi），我们测试了PTC52是否参与控制pPORA导入和Pchlide稳态。如下所示，缺乏转录本和PTC52蛋白的植物无法导入pPORA，并因绿化过程中Pchlide积累过多导致单线态氧形成而死亡。在遗传学研究中，可能由于胚胎致死性而无法获得纯合敲除突变体，这表明PTC52在植物胚胎的初始绿化中起作用。系统发育研究在单细胞光合细菌和高等植物中鉴定出了类似PTC52的基因，这表明与PTC52相关的生化功能可能具有古老的进化起源。PTC52还具有与细菌加氧酶保守的基序，如来自[具体来源未提及]的3-酮类固醇9-α-羟化酶（KshA）的末端加氧酶成分。PTC52结构的三维建模允许预测有助于该酶底物特异性的氨基酸残基。使用定点诱变来测试预测的PTC52模型，并深入了解这种 Rieske 非血红素加氧酶的反应机制。
