Identifying esterase and lipase activity in proteins of unknown function.

Abstract

Over 3800 structures in the Protein Data Bank (PDB) have unknown function. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) curriculum uses authentic inquiry to teach students to use structural bioinformatics tools to compare these structures to known enzymes and predict a function. We have predicted esterase or lipase function for several structures after performing both global and local structure alignments to identify similar structures in the PDB as well as specific amino acids that may be active site motifs. This function was supported by sequence alignment data from BLAST and PFam. SwissDock was then used for ligand docking to determine ester and lipid substrates to use for testing activity. Students in the biochemistry lab next used standard wet-lab biochemistry techniques to express and purify the target enzymes and performed kinetic assays using p-nitrophenyl acetate to test for esterase activity. After completing the BASIL curriculum, students continued with independent research projects to develop assays to test for lipase function. We have focused our efforts on the protein structures with PDB IDs 2O14 and 4Q7Q, as they showed the most promise through initial enzymatic testing. Both proteins show significant esterase activity in hydrolyzing both p-nitrophenyl acetate and p-nitrophenyl butyrate. Additionally, both proteins are able to hydrolyze p-nitrophenyl dodecanoate when sodium deoxycholate is present in the buffer solution for solubility of the substrate. We have also incorporated the p-nitrophenyl dodecanoate in DMPC vesicles, and testing of substrate hydrolysis with these vesicles is underway. We have also tested pancreatic lipase under all these conditions as a positive control for the experiments. Students will be creating a Proteopedia page for each protein. This project has been supported in part by NSF IUSE 1709355 and 2141908.