Buckley David P, Becker Donald F, Tanner John J
Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.
Department of Biochemistry, Redox Biology Center, University of Nebraska, Lincoln, Nebraska, USA.
J Biol Chem. 2025 Jul 28;301(9):110532. doi: 10.1016/j.jbc.2025.110532.
The bifunctional enzyme proline utilization A (PutA) catalyzes the two-step oxidation of L-proline to L-glutamate using proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase (GSALDH) domains. The two active sites are 42 Å apart and connected by a buried tunnel that is hypothesized to channel the intermediates Δ-pyrroline-5-carboxylate (P5C) and/or L-glutamate-γ-semialdehyde (GSAL). Kinetic and conventional X-ray crystallography of PutA from Sinorhizobium meliloti (SmPutA) were used to capture high resolution (1.47-1.88 Å) structures of states along the catalytic cycle, including a novel FADH-proline covalent adduct in the PRODH site, the intermediate P5C bound noncovalently in the reduced PRODH active site, the covalent acyl-enzyme intermediate of the GSALDH reaction, and noncovalent complexes of GSAL and the final product L-glutamate in the GSALDH active site. The FADH-proline covalent adduct resembles a stable species predicted from quantum mechanical electronic structure calculations of the PRODH reaction. The GSALDH domain complexes are consistent with conservation of substrate recognition and catalytic mechanism by the aldehyde dehydrogenase superfamily. The structure of reduced SmPutA with the P5C bound in the PRODH active site was used as the starting point for molecular dynamics simulations (21 × 2 μs trajectories). P5C diffuses from the PRODH active site into the tunnel in most of the trajectories, but rarely dissociates completely from the enzyme, consistent with previous kinetic evidence of a substrate channeling mechanism. The simulations also provide insight into protein conformational changes associated with substrate channeling, including the opening and closing of a conserved ion pair gate.
双功能酶脯氨酸利用A（PutA）利用脯氨酸脱氢酶（PRODH）和L-谷氨酸-γ-半醛脱氢酶（GSALDH）结构域催化L-脯氨酸两步氧化为L-谷氨酸。两个活性位点相距42 Å，由一条埋藏的通道相连，据推测该通道可引导中间体Δ-吡咯啉-5-羧酸（P5C）和/或L-谷氨酸-γ-半醛（GSAL）。利用苜蓿中华根瘤菌（SmPutA）的PutA的动力学和传统X射线晶体学来捕获催化循环中各状态的高分辨率（1.47 - 1.88 Å）结构，包括PRODH位点中一种新型的FADH-脯氨酸共价加合物、在还原的PRODH活性位点中非共价结合的中间体P5C、GSALDH反应的共价酰基酶中间体，以及GSALDH活性位点中GSAL和最终产物L-谷氨酸的非共价复合物。FADH-脯氨酸共价加合物类似于从PRODH反应的量子力学电子结构计算预测的稳定物种。GSALDH结构域复合物与醛脱氢酶超家族对底物识别和催化机制的保守性一致。将P5C结合在PRODH活性位点的还原型SmPutA结构用作分子动力学模拟（21×2 μs轨迹）的起点。在大多数轨迹中，P5C从PRODH活性位点扩散到通道中，但很少完全从酶上解离，这与先前底物通道化机制的动力学证据一致。模拟还深入了解了与底物通道化相关的蛋白质构象变化，包括一个保守离子对门的打开和关闭。
