Data_Sheet_1_Enzymology and Regulation of δ1-Pyrroline-5-Carboxylate Synthetase 2 From Rice.PDF

Under several stress conditions, such as excess salt and drought, many plants accumulate proline inside the cell, which is believed to help counteracting the adverse effects of low water potential. This increase mainly relies upon transcriptional induction of δ1-pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in proline biosynthesis from glutamate. P5CS mediates both the phosphorylation of glutamate and the reduction of γ-glutamylphosphate to glutamate-5-semialdehyde, which spontaneously cyclizes to δ1-pyrroline-5-carboxylate (P5C). In most higher plants, two isoforms of P5CS have been found, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate the regulation of P5CS at the transcriptional level, to date, the properties of the enzyme have been only poorly studied. As a consequence, the descriptions of post-translational regulatory mechanisms have largely been limited to feedback-inhibition by proline. Here, we report cloning and heterologous expression of P5CS2 from Oryza sativa. The protein has been fully characterized from a functional point of view, using an assay method that allows following the physiological reaction of the enzyme. Kinetic analyses show that the activity is subjected to a wide array of regulatory mechanisms, ranging from product inhibition to feedback inhibition by proline and other amino acids. These findings confirm long-hypothesized influences of both, the redox status of the cell and nitrogen availability, on proline biosynthesis.

在高盐、干旱等多种胁迫条件下，多数植物会在细胞内积累脯氨酸，这被认为有助于抵消低水势带来的不利影响。该积累过程主要依赖于δ1-吡咯啉-5-羧酸合成酶（δ1-pyrroline-5-carboxylate synthetase，P5CS）的转录诱导——该酶催化以谷氨酸为底物的脯氨酸生物合成前两步反应。P5CS既可介导谷氨酸的磷酸化反应，也可催化γ-谷氨酰磷酸还原为谷氨酸-5-半醛，后者可自发环化形成δ1-吡咯啉-5-羧酸（P5C）。在多数高等植物中已发现两种P5CS同工酶：一种为组成型表达，以满足蛋白质合成所需的脯氨酸需求；另一种则受胁迫诱导表达。尽管已有诸多研究探讨P5CS的转录调控机制，但截至目前，对该酶的酶学性质的研究仍较为匮乏。因此，目前关于其翻译后调控机制的研究大多仅局限于脯氨酸的反馈抑制作用。本研究成功克隆了水稻（Oryza sativa）的P5CS2基因并实现其异源表达，采用可追踪该酶生理反应的检测方法，从功能层面完成了该蛋白的全面表征。动力学分析结果表明，该酶的活性受到多种调控机制的影响，涵盖产物抑制以及脯氨酸与其他氨基酸的反馈抑制等。本研究结果证实了此前长期以来的假说，即细胞氧化还原状态与氮素有效性均可对脯氨酸生物合成产生调控作用。