Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺-dependent oxidations of L-glutamate-γ-semialdehyde to L-glutamate (proline metabolism), and 4-hydroxy-L-glutamate-γ-semialdehyde to 4-erythro-hydroxy-L-glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4-hydroxyproline using steady-state kinetics and X-ray crystallography. Trans-4-hydroxy-L-proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L-proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L-stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans-4-hydroxy-L-proline and trans-4-hydroxy-D-proline. The structures suggest that the 10-fold greater preference for the L-stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans-4-hydroxy-L-proline. In contrast, the amine group of the D-stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans-4-hydroxy-L-proline, analogous to the known inhibition of proline catabolism by L-proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans-4-hydroxy-L-proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.

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反式4-羟基-L-脯氨酸立体特异性抑制脯氨酸/羟脯氨酸分解代谢酶ALDH4A1的结构基础

醛脱氢酶 4A1 (ALDH4A1) 催化脯氨酸和羟脯氨酸分解代谢的最后步骤。它是一种双底物酶，可催化 NAD ⁺L-谷氨酸-γ-半醛氧化成L-谷氨酸（脯氨酸代谢），4-羟基-L-谷氨酸-γ-半醛氧化成4-赤-羟基-L-谷氨酸（羟脯氨酸代谢）。在这里，我们使用稳态动力学和 X 射线晶体学研究了脯氨酸和 4-羟基脯氨酸的六种立体异构体对小鼠 ALDH4A1 的抑制作用。Trans-4-hydroxy-L-proline 是所研究的最强抑制剂，其特点是竞争性抑制常数为 0.7 mM，其次是 L-proline (1.9 mM)。其他化合物是非常弱的抑制剂（大约 10 mM 或更大）。通过解析与反式-4-羟基-L-脯氨酸和反式-4-羟基-D-脯氨酸复合的 ALDH4A1 的晶体结构，可以深入了解 L-立体异构体的选择性。这些结构表明，对 L-立体异构体的 10 倍更大的偏好是由于丝氨酸残基与反式-4-羟基-L-脯氨酸的胺基形成氢键。相反，由于吡咯烷环的不同方向，D-立体异构体的胺基缺乏与酶的直接相互作用。这些结果表明羟脯氨酸分解代谢受到反式4-羟基-L-脯氨酸的底物抑制，类似于已知的L-脯氨酸对脯氨酸分解代谢的抑制。此外，靶向羟脯氨酸分解代谢的第一种酶的药物，通过提高反式-4-羟基-L-脯氨酸的水平，可能会通过抑制 ALDH4A1 无意中损害脯氨酸分解代谢。由于吡咯烷环的不同方向，D-立体异构体的胺基缺乏与酶的直接相互作用。这些结果表明羟脯氨酸分解代谢受到反式4-羟基-L-脯氨酸的底物抑制，类似于已知的L-脯氨酸对脯氨酸分解代谢的抑制。此外，靶向羟脯氨酸分解代谢的第一种酶的药物，通过提高反式-4-羟基-L-脯氨酸的水平，可能会通过抑制 ALDH4A1 无意中损害脯氨酸分解代谢。由于吡咯烷环的不同方向，D-立体异构体的胺基缺乏与酶的直接相互作用。这些结果表明羟脯氨酸分解代谢受到反式4-羟基-L-脯氨酸的底物抑制，类似于已知的L-脯氨酸对脯氨酸分解代谢的抑制。此外，靶向羟脯氨酸分解代谢的第一种酶的药物，通过提高反式-4-羟基-L-脯氨酸的水平，可能会通过抑制 ALDH4A1 无意中损害脯氨酸分解代谢。