Elucidating the Function of 3L1W, a Functionally Unknown Enzyme from Enterococcus faecalis V583
Abstract
As of February 2022, there were 4057 protein structures with unknown functions in the Protein Data Bank. In all biological organisms there are a variety of enzymes that speed up key biochemical pathways. Despite having a known sequence and structure, many enzymes remain uncharacterized. One relevant enzyme identified is 3L1W, whose function has yet to be elucidated. The P.O.I. was isolated and imaged via Xray crystallography from the vancomycin resistant strain E. faecalis V583.
The purpose of this research was to provide an accurate characterization of 3L1W and its substrates, thus directing our approach of combining in silico and in vitro techniques. Protein visualization and structural alignments were conducted via PyMOL, BLAST, and Pfam, while structural alignments and ligand docking utilized Dali and PyRX software. We predicted the function of the protein and identify its most promising substrates. Confirmation of the enzyme’s function can be developed through protein transformation, expression, and purification protocols. 3L1W was transformed and expressed in E. coli cells and isolated. The activity of 3L1W was measured for the substrate 4-nitrophenyl acetate, and enzyme kinetic hydrolase data was obtained. Based on 3L1W’s hydrolase activity and the function of its homologs, a topoisomerase assay was performed. Together these tools can help identify future wet lab experiments to conduct, as well as continue to elucidate further the function of unknown proteins. Additionally, understanding the function of 3L1W has large implications on better understanding the human GI tract and human antibiotic resistance.
Faculty Sponsors
Dr. Arthur Sikora
Project Type
Event
Location
Alvin Sherman Library
Start Date
4-6-2022 12:00 PM
End Date
4-7-2022 5:00 PM
Elucidating the Function of 3L1W, a Functionally Unknown Enzyme from Enterococcus faecalis V583
Alvin Sherman Library
As of February 2022, there were 4057 protein structures with unknown functions in the Protein Data Bank. In all biological organisms there are a variety of enzymes that speed up key biochemical pathways. Despite having a known sequence and structure, many enzymes remain uncharacterized. One relevant enzyme identified is 3L1W, whose function has yet to be elucidated. The P.O.I. was isolated and imaged via Xray crystallography from the vancomycin resistant strain E. faecalis V583.
The purpose of this research was to provide an accurate characterization of 3L1W and its substrates, thus directing our approach of combining in silico and in vitro techniques. Protein visualization and structural alignments were conducted via PyMOL, BLAST, and Pfam, while structural alignments and ligand docking utilized Dali and PyRX software. We predicted the function of the protein and identify its most promising substrates. Confirmation of the enzyme’s function can be developed through protein transformation, expression, and purification protocols. 3L1W was transformed and expressed in E. coli cells and isolated. The activity of 3L1W was measured for the substrate 4-nitrophenyl acetate, and enzyme kinetic hydrolase data was obtained. Based on 3L1W’s hydrolase activity and the function of its homologs, a topoisomerase assay was performed. Together these tools can help identify future wet lab experiments to conduct, as well as continue to elucidate further the function of unknown proteins. Additionally, understanding the function of 3L1W has large implications on better understanding the human GI tract and human antibiotic resistance.
