Structure–function relationships of the 5‐oxoprolinase subunit A: Guiding biological sciences students down the path less traveled

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.