Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, USA.
Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2878-83. doi: 10.1073/pnas.0906101107. Epub 2010 Feb 1.
The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD(+)-dependent Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 A resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 A. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 A and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Delta(1)-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Delta(1)-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.
双功能脯氨酸分解黄素酶，脯氨酸利用 A（PutA），通过 FAD 依赖性脯氨酸脱氢酶（PRODH）和 NAD（+）依赖性 Delta（1）-吡咯啉-5-羧酸脱氢酶（P5CDH）结构域的顺序活性，催化脯氨酸氧化为谷氨酸。尽管已经知道 PutA 的一些结构域的结构，但以前尚未解决全长蛋白的结构。在这里，我们报告了来自大豆根瘤菌的 PutA 的 2.1Å分辨率晶体结构，以及来自小角度 X 射线散射、分析超速离心、稳态和快速反应动力学的数据。PutA 在溶液中形成一个直径为 150Å的环形四聚体。在每个原蛋白中，PRODH 和 P5CDH 活性位点彼此面对，距离为 41Å，并通过一个大的、不规则形状的腔连接。动力学测量表明，谷氨酸的产生没有滞后阶段，这表明中间产物 Delta（1）-吡咯啉-5-羧酸更倾向于转移到 P5CDH 结构域，而不是释放到介质中。结构和动力学数据表明，该腔既是用于将 Delta（1）-吡咯啉-5-羧酸水解为谷氨酸半醛的微观容器，也是将谷氨酸半醛运输到 P5CDH 活性位点的受保护通道。
