Bioinformatics was recently introduced as a module for both undergraduate and postgraduate biological sciences students at our institution. Our experience shows that inquiry‐based hands‐on exercises provide the most efficient approach to bioinformatic straining. In this article, we report a structural bioinformatics project carried out by Master degree students to determine structure–function relationships of the uncharacterized prokaryotic 5‐oxoprolinase subunit A (PxpA). PxpA associates with the PxpBC complex to form a functional 5‐oxoprolinase enzyme for conversion of 5‐oxoproline to L‐glutamate. Although the exact role of PxpA is yet to be determined, it has been demonstrated that PxpBC catalyses the first step of the reaction, which is phosphorylation of 5‐oxoproline. Here, we provide evidence that PxpA is involved in the last two steps of the reaction:decyclization of the labile phosphorylated 5‐oxoproline to the equally labile γ‐glutamylphosphate, and subsequent dephosphorylation to L‐glutamate. Structural bioinformatics analysis of four putative PxpA structures revealed that PxpA adopts a non‐canonical TIM barrel fold with well‐characterized TIM barrel enzyme features. These include a C‐terminal groove comprising potentially essential conserved amino acid residues organized into putative motifs. Phylogenetic analysis suggests a relationship between taxonomic grouping and PxpA oligomerization. PxpA forms a tunnel upon ligand binding, thus suggesting that the PxpABC complex employs the mechanism of substrate channeling to protect labile intermediates. Ultimately, students were able to form a testable hypothesis on the function of PxpA, an achievement we consider encouraging other students to emulate. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):620–631, 2019.

中文翻译：

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5-oxoprolinase 亚基 A 的结构-功能关系：引导生物科学学生走少有人迹的道路

生物信息学最近被引入作为我们机构本科生和研究生生物科学学生的模块。我们的经验表明，基于探究的动手练习为生物信息学应变提供了最有效的方法。在本文中，我们报告了由硕士生进行的结构生物信息学项目，以确定未表征的原核 5-氧代脯氨酸酶亚基 A (PxpA) 的结构-功能关系。PxpA 与 PxpBC 复合物结合形成功能性 5-氧脯氨酸酶，用于将 5-氧脯氨酸转化为 L-谷氨酸。尽管 PxpA 的确切作用尚未确定，但已证明 PxpBC 催化反应的第一步，即 5-氧代脯氨酸的磷酸化。这里，我们提供的证据表明 PxpA 参与了反应的最后两个步骤：不稳定的磷酸化 5-氧代脯氨酸脱环为同样不稳定的 γ-谷氨酰磷酸，然后去磷酸化为 L-谷氨酸。四种假定的 PxpA 结构的结构生物信息学分析表明，PxpA 采用具有良好表征的 TIM 桶状酶特征的非规范 TIM 桶状折叠。这些包括一个 C 端凹槽，其中包含组织成推定基序的潜在必需的保守氨基酸残基。系统发育分析表明分类分组和 PxpA 寡聚化之间存在关系。PxpA 在配体结合后形成隧道，因此表明 PxpABC 复合物采用底物通道机制来保护不稳定的中间体。最终，学生能够对 PxpA 的功能形成可检验的假设，我们认为这是鼓励其他学生效仿的成就。© 2019 国际生物化学与分子生物学联盟，47(6):620–631，2019。