Departments of Chemistry and Biochemistry, University of Missouri, Columbia, MO 65211.
Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):3389-94. doi: 10.1073/pnas.1321621111. Epub 2014 Feb 18.
Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of L-proline to L-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004-residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å-long tunnel that links the two active sites and six smaller tunnels that connect the main tunnel to the bulk medium. The locations of these tunnels and their responses to ligand binding and flavin reduction suggest hypotheses about how proline, water, and quinones enter the tunnel system and where L-glutamate exits. Kinetic measurements show that glutamate production from proline occurs without a lag phase, consistent with substrate channeling and implying that the observed tunnel is functionally relevant. Furthermore, the structure of reduced PutA complexed with menadione bisulfite reveals the elusive quinone-binding site. The benzoquinone binds within 4.0 Å of the flavin si face, consistent with direct electron transfer. The location of the quinone site implies that the concave surface of the PutA dimer approaches the membrane. Altogether, these results provide insight into how PutAs couple proline oxidation to quinone reduction.
脯氨酸利用 A（PutA）蛋白是具有双功能的外周膜黄素酶，通过脯氨酸脱氢酶和醛脱氢酶结构域的顺序活性，催化 L-脯氨酸氧化为 L-谷氨酸。位于革兰氏阴性菌的内膜上，PutA 通过将从环境中导入的脯氨酸氧化与膜相关醌的还原偶联，在能量代谢中发挥主要作用。在这里，我们报告了来自 Geobacter sulfurreducens 的 1004 个残基的 PutA 的七个晶体结构，同时通过小角度 X 射线散射确定了蛋白质的寡聚状态，并对底物通道化和醌还原的动力学特性进行了表征。这些结构揭示了一个精心设计且动态的隧道系统，其特征是一条 75 Å 长的隧道，将两个活性位点连接起来，还有六条较小的隧道将主隧道与主体介质连接起来。这些隧道的位置及其对配体结合和黄素还原的响应提出了关于脯氨酸、水和醌如何进入隧道系统以及 L-谷氨酸从何处排出的假说。动力学测量表明，脯氨酸生成谷氨酸没有滞后期，这与底物通道化一致，意味着观察到的隧道在功能上是相关的。此外，与亚硫酸甲萘醌结合的还原型 PutA 结构揭示了难以捉摸的醌结合位点。苯醌在黄素 si 面的 4.0 Å 内结合，与直接电子转移一致。醌结合位点的位置表明 PutA 二聚体的凹面接近膜。总之，这些结果提供了对 PutA 如何将脯氨酸氧化与醌还原偶联的深入了解。
