Proline dehydrogenase (ProDH) is a flavoenzyme that catalyzes the oxidation of proline (Pro) into Δ1-pyrroline-5-carboxylate (P5C). In eukaryotes, ProDH coordinates with different Pro metabolism enzymes to control energy supply or stress responses signaling. Heterologous expression and crystallization of prokaryotic enzymes provided key data on their active center, folding capacity and oligomerization status. In contrast, eukaryotic ProDHs have not been crystallized so far, and their study as recombinant proteins remains limited. Plants contain two isoforms of ProDH with non-redundant functions. To contribute to the study of these enzymes, we describe the modeling, expression in E. coli, purification, and characterization of the Arabidopsis isoenzymes, AtProDH1 and AtProDH2. The 3D model suggested that both proteins adopt a distorted barrel structure (βα) with a cap formed by N-terminal α helices. The expression of two types of N-terminal deletion proteins indicated that this domain affected enzyme activity. Full-length enzymes had Km values similar to those of native proteins, whereas truncated proteins were inactive. Moreover, the first α helix proved to be necessary for AtProDH1 and AtProDH2 activities. Interestingly, both isoenzymes were able to oligomerize and this also required the first N-terminal α helix. Thus, we report the first insights into structure-function relationship of plant ProDHs demonstrating that the N-terminus, although not directly involved in catalysis, controls enzyme arrangement and activity. The resources generated here could be useful to analyze other plant ProDH features, such as its coordination with other enzymes, and differences between ProDH1 and ProDH2, providing new information on its effects on stress tolerance.

脯氨酸脱氢酶（ProDH）是一种黄素酶，可催化脯氨酸（Pro）氧化为Δ1-吡咯啉-5-羧酸盐（P5C）。在真核生物中，ProDH与不同的Pro代谢酶协同作用，以控制能量供应或应激反应信号传导。原核酶的异源表达和结晶提供了有关其活性中心，折叠能力和低聚状态的关键数据。相反，到目前为止，真核ProDHs尚未结晶，其作为重组蛋白的研究仍然有限。植物包含具有非冗余功能的ProDH的两个同工型。为了有助于这些酶的研究，我们描述了在大肠杆菌中的建模，表达，拟南芥同工酶AtProDH1和AtProDH2的纯化，表征。3D模型表明这两种蛋白质均采用扭曲的桶状结构（βα），其帽盖由N端α螺旋形成。两种类型的N末端缺失蛋白的表达表明该结构域影响酶的活性。全长酶的Km值与天然蛋白相似，而截短的蛋白则无活性。此外，第一个α螺旋被证明对于AtProDH1和AtProDH2活动是必需的。有趣的是，两种同工酶都能够寡聚，这也需要第一个N末端α螺旋。因此，我们报道植物ProDHs的结构-功能关系的第一个见解，表明N末端虽然不直接参与催化，但控制酶的排列和活性。