Functional Characterization of 2QRU Based on Structural and Enzymatic Analysis

Abstract

Beginning in 2000, the National Institutes of Health's Protein Structure Initiative was a fifteen‐year program that worked to solve the structures of many proteins with known DNA sequences. The successful initiative yielded the publication of more than 500 previously uncharacterized proteins. The present study analyzes one such protein, known as 2QRU, a predicted alpha/beta hydrolase found in Enterococcus faecalis. Using modules outlined by BASIL (Biochemistry Authentic Scientific Inquiry Lab) and resources available in a high school molecular biology teaching laboratory, the specific objective of this study is to functionally characterize 2QRU based on structural analysis of the protein. In silico enzyme characterization for 2QRU was carried out using BLAST, Pfam, and Dali servers, followed by active site analysis using the PyMOL plug‐in ProMOL. ProMOL generated a list of proteins with active sites that were structurally similar to 2QRU. For each of the hits with a Levenshtein distance of zero, the predicted catalytic triad for 2QRU was composed of a Ser‐102, Asp‐219, and His‐247. Secondary structure alignments using PROMALS3D and structural alignments in PyMOL were used to compare 2QRU to the active site matches identified using ProMOL. This structural analysis suggests PDB entries 3LIP and 1YSC ‐ a triacylglycerol lipase and serine carboxypeptidase, respectively ‐ are most similar to 2QRU and share a conserved catalytic triad. However, serine carboxypeptidases have yet to be discovered in bacteria, indicating 2QRU may function as a lipase. To investigate the lipase activity of 2QRU in vitro, recombinant his‐tagged protein was isolated from E. coli. Subsequent enzymatic assays using spectrophotometric analysis will be carried out based on methods described for the characterization of Pseudomonas cepacia lipase (PCL) (PDB ID 3LIP) using the substrate p‐Nitrophenyl palmitate, an ester of palmitic acid. As a research project in a secondary school molecular biology research course, this study not only contributes to the field of bacterial lipase research, but also offers students an authentic research experience at an early stage in their scientific careers.Support or Funding InformationThis work was funded by The Pingry School.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.