Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of l-proline to Δ1-pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analogue thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH. Structures of the bifunctional proline catabolic enzyme proline utilization A (PutA) determined from crystals grown in the presence of T2C feature strong electron density for a 5-membered ring species resembling l-T2C covalently bound to the N5 of the FAD in the PRODH domain. The modified FAD exhibits a large butterfly bend angle, indicating that the FAD is locked into the 2-electron reduced state. Reduction of the FAD is consistent with the crystals lacking the distinctive yellow color of the oxidized enzyme and stopped-flow kinetic data showing that T2C is a substrate for the PRODH domain of PutA. A mechanism is proposed in which PRODH catalyzes the oxidation of T2C at the C atom adjacent to the S atom of the thiazolidine ring (C5). Then, the N5 atom of the reduced FAD attacks the C5 of the oxidized T2C species, resulting in the covalent adduct observed in the crystal structure. To our knowledge, this is the first report of T2C inactivating (or inhibiting) PRODH or any other flavoenzyme. These results may inform the design of new mechanism-based inactivators of PRODH for use as chemical probes to study the roles of proline metabolism in cancer.

中文翻译：

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噻唑烷-2-羧酸盐对脯氨酸脱氢酶中黄素的共价修饰。

脯氨酸脱氢酶（PRODH）催化脯氨酸分解代谢的第一步，即L-脯氨酸的FAD依赖性2电子氧化为Δ1-吡咯啉-5-羧酸酯。PRODH已经成为可能的癌症治疗靶标，因此对PRODH的抑制是令人关注的。在这里，我们显示脯氨酸类似物噻唑烷-2-羧酸盐（T2C）是PRODH的一种基于机理的灭活剂。由在T2C存在下生长的晶体确定的双功能脯氨酸分解代谢酶脯氨酸利用率A（PutA）的结构具有5电子环结构的强电子密度，类似于在PRODH域中共价键合至FAD N5的1-T2C。修改后的FAD表现出较大的蝶形弯曲角度，表明FAD锁定为2电子还原态。FAD的减少与缺乏氧化酶显着黄色的晶体一致，并且停流动力学数据表明T2C是PutA PRODH域的底物。提出了一种机制，其中PRODH催化与噻唑烷环（C5）的S原子相邻的C原子处的T2C氧化。然后，还原的FAD的N5原子攻击氧化的T2C物种的C5，导致在晶体结构中观察到共价加合物。据我们所知，这是T2C灭活（或抑制）PRODH或任何其他黄素酶的第一个报道。这些结果可能有助于设计新的基于机制的PRODH灭活剂，用作化学探针以研究脯氨酸在癌症中的作用。