Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of l-proline to l-glutamate using spatially-separated proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH, a.k.a. ALDH4A1) active sites. The observation that l-proline inhibits both the GSALDH activity of PutA and monofunctional GSALDHs motivated us to study the inhibition of PutA by proline stereoisomers and analogs. Here we report five high-resolution crystal structures of PutA with the following ligands bound in the GSALDH active site: d-proline, trans-4-hydroxy-d-proline, cis-4-hydroxy-d-proline, l-proline, and trans-4-hydroxy-l-proline. Three of the structures are of ternary complexes of the enzyme with an inhibitor and either NAD⁺ or NADH. To our knowledge, the NADH complex is the first for any GSALDH. The structures reveal a conserved mode of recognition of the inhibitor carboxylate, which results in the pyrrolidine rings of the d- and l-isomers having different orientations and different hydrogen bonding environments. Activity assays show that the compounds are weak inhibitors with millimolar inhibition constants. Curiously, although the inhibitors occupy the aldehyde binding site, kinetic measurements show the inhibition is uncompetitive. Uncompetitive inhibition may involve proline binding to a remote site or to the enzyme-NADH complex. Together, the structural and kinetic data expand our understanding of how proline-like molecules interact with GSALDH, reveal insight into the relationship between stereochemistry and inhibitor affinity, and demonstrate the pitfalls of inferring the mechanism of inhibition from crystal structures alone.

脯氨酸利用 A (PutA) 蛋白是双功能脯氨酸分解代谢酶，使用空间分离的脯氨酸脱氢酶和l-谷氨酸-γ-半醛脱氢酶 (GSALDH，又名 ALDH4A1) 活性位点催化l-脯氨酸的 4 电子氧化为l-谷氨酸. l -脯氨酸抑制PutA和单功能GSALDH的GSALDH活性的观察促使我们研究脯氨酸立体异构体和类似物对PutA的抑制。在这里，我们报告了 PutA 的五种高分辨率晶体结构，以下配体结合在 GSALDH 活性位点：d-脯氨酸、反式-4-羟基-d-脯氨酸、顺式-4-羟基-d-脯氨酸、l-脯氨酸和反式-4-羟基-l-脯氨酸。其中三个结构是酶与抑制剂和 NAD ⁺或 NADH 的三元复合物。据我们所知，NADH 复合体是任何 GSALDH 的第一个复合体。这些结构揭示了一种识别抑制剂羧酸盐的保守模式，这导致了d - 和l的吡咯烷环-具有不同取向和不同氢键环境的异构体。活性测定表明，这些化合物是具有毫摩尔抑制常数的弱抑制剂。奇怪的是，虽然抑制剂占据了醛结合位点，但动力学测量显示抑制是非竞争性的。非竞争性抑制可能涉及脯氨酸与远程位点或酶-NADH 复合物的结合。总之，结构和动力学数据扩展了我们对类脯氨酸分子如何与 GSALDH 相互作用的理解，揭示了立体化学和抑制剂亲和力之间的关系，并证明了仅从晶体结构推断抑制机制的缺陷。