Testing for Lipase Activity in Enzymes of Unknown Function

Abstract

There are over 3800 structures in the Protein Data Bank (PDB) that 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 lipase function for several structures after performing both global and local structure alignments using Dali and ProMOL. This function was supported by sequence alignment data from BLAST and PFam. Autodock Vina and PyRx were then used for ligand docking to determine possible lipid substrates to test activity. Students next used standard wet-lab biochemistry techniques to express and purify the target enzymes and performed kinetic assays first 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 with the p-nitrophenyl acetate substrate. Additionally, both proteins show significant hydrolysis of 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 both proteins can hydrolyze the substrate from the vesicle. We have tested pancreatic lipase under all these conditions as a positive control for the experiments. Students will eventually be able to update the PDB with the demonstrated function for these proteins, and they will also create a Proteopedia page for each protein. This project is supported in part by NSF IUSE 1709355.