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.

在多种压力条件下，例如过量盐分和干旱，许多植物会在细胞内积累脯氨酸，这被认为有助于抵消低水势的不利影响。这种增加主要是依赖于δ的转录诱导¹吡咯啉-5-羧酸合成酶（P5CS），其催化从谷氨酸生物合成脯氨酸的前两个步骤的酶。P5CS 介导谷氨酸的磷酸化和 γ-谷氨酰磷酸还原为谷氨酸 5-半醛，后者自发环化为 δ ¹-pyrroline-5-carboxylate (P5C)。在大多数高等植物中，已发现 P5CS 的两种同工型，一种是组成型表达以满足蛋白质合成对脯氨酸的需求，另一种是胁迫诱导的。尽管有大量论文在转录水平上研究 P5CS 的调控，但迄今为止，对该酶的特性研究甚少。因此，对翻译后调节机制的描述主要限于脯氨酸的反馈抑制。在这里，我们报告了 P5CS2 的克隆和异源表达水稻. 从功能的角度来看，该蛋白质已被完全表征，使用的分析方法允许跟踪酶的生理反应。动力学分析表明，该活性受到一系列调节机制的影响，从产物抑制到脯氨酸和其他氨基酸的反馈抑制。这些发现证实了长期假设的细胞氧化还原状态和氮可用性对脯氨酸生物合成的影响。