Korasick David A, Singh Harkewal, Pemberton Travis A, Luo Min, Dhatwalia Richa, Tanner John J
Department of Biochemistry, University of Missouri, Columbia, MO, USA.
Department of Chemistry, University of Missouri, Columbia, MO, USA.
FEBS J. 2017 Sep;284(18):3029-3049. doi: 10.1111/febs.14165. Epub 2017 Aug 1.
Many enzymes form homooligomers, yet the functional significance of self-association is seldom obvious. Herein, we examine the connection between oligomerization and catalytic function for proline utilization A (PutA) enzymes. PutAs are bifunctional enzymes that catalyze both reactions of proline catabolism. Type A PutAs are the smallest members of the family, possessing a minimal domain architecture consisting of N-terminal proline dehydrogenase and C-terminal l-glutamate-γ-semialdehyde dehydrogenase modules. Type A PutAs form domain-swapped dimers, and in one case (Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-shaped tetramer. Whereas the dimer has a clear role in substrate channeling, the functional significance of the tetramer is unknown. To address this question, we performed structural studies of four-type A PutAs from two clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus PutA covalently inactivated by N-propargylglycine revealed a fold and substrate-channeling tunnel similar to other PutAs. Small-angle X-ray scattering (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio PutA is dimeric in solution, in contrast to the prediction from crystal packing of a stable tetrameric assembly. SAXS studies of two other type A PutAs from separate clades also suggested that the dimer predominates in solution. To assess whether the tetramer of B. japonicum PutA is necessary for catalytic function, a hot spot disruption mutant that cleanly produces dimeric protein was generated. The dimeric variant exhibited kinetic parameters similar to the wild-type enzyme. These results implicate the domain-swapped dimer as the core structural and functional unit of type A PutAs.
Proline dehydrogenase (EC 1.5.5.2); l-glutamate-γ-semialdehyde dehydrogenase (EC 1.2.1.88).
The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession number 5UR2. The SAXS data have been deposited in the SASBDB under the following accession codes: SASDCP3 (BbPutA), SASDCQ3 (DvPutA 1.5 mg·mL ), SASDCX3 (DvPutA 3.0 mg·mL ), SASDCY3 (DvPutA 4.5 mg·mL ), SASDCR3 (LpPutA 3.0 mg·mL ), SASDCV3 (LpPutA 5.0 mg·mL ), SASDCW3 (LpPutA 8.0 mg·mL ), SASDCS3 (BjPutA 2.3 mg·mL ), SASDCT3 (BjPutA 4.7 mg·mL ), SASDCU3 (BjPutA 7.0 mg·mL ), SASDCZ3 (R51E 2.3 mg·mL ), SASDC24 (R51E 4.7 mg·mL ), SASDC34 (R51E 7.0 mg·mL ).
许多酶形成同型寡聚体，然而自我缔合的功能意义却很少是显而易见的。在此，我们研究脯氨酸利用A（PutA）酶的寡聚化与催化功能之间的联系。PutA酶是催化脯氨酸分解代谢两个反应的双功能酶。A型PutA是该家族中最小的成员，具有由N端脯氨酸脱氢酶和C端L-谷氨酸-γ-半醛脱氢酶模块组成的最小结构域架构。A型PutA形成结构域交换二聚体，在一个例子中（日本慢生根瘤菌PutA），两个二聚体组装成环形四聚体。虽然二聚体在底物通道化中具有明确作用，但四聚体的功能意义尚不清楚。为了解决这个问题，我们对来自PutA树两个进化枝的四种A型PutA进行了结构研究。被N-炔丙基甘氨酸共价灭活的食菌蛭弧菌PutA的晶体结构揭示了与其他PutA相似的折叠和底物通道化隧道。小角X射线散射（SAXS）和分析超速离心表明，与稳定四聚体组装的晶体堆积预测相反，食菌蛭弧菌PutA在溶液中是二聚体。对来自不同进化枝的另外两种A型PutA的SAXS研究也表明二聚体在溶液中占主导。为了评估日本慢生根瘤菌PutA的四聚体对于催化功能是否必要，产生了一个能清晰产生二聚体蛋白的热点破坏突变体。二聚体变体表现出与野生型酶相似的动力学参数。这些结果表明结构域交换二聚体是A型PutA的核心结构和功能单元。
脯氨酸脱氢酶（EC 1.5.5.2）；L-谷氨酸-γ-半醛脱氢酶（EC 1.2.1.88）。
原子坐标和结构因子振幅已存入蛋白质数据库，登录号为5UR2。SAXS数据已存入SASBDB，登录代码如下：SASDCP3（BbPutA）、SASDCQ3（DvPutA 1.5 mg·mL）、SASDCX3（DvPutA 3.0 mg·mL）、SASDCY3（DvPutA 4.5 mg·mL）、SASDCR3（LpPutA 3.0 mg·mL）、SASDCV3（LpPutA 5.0 mg·mL）、SASDCW3（LpPutA 8.0 mg·mL）、SASDCS3（BjPutA 2.3 mg·mL）、SASDCT3（BjPutA 4.7 mg·mL）、SASDCU3（BjPutA 7.0 mg·mL）、SASDCZ3（R51E 2.3 mg·mL）、SASDC24（R51E 4.7 mg·mL）、SASDC34（R51E 7.0 mg·mL）。
