Substrate Specificity of the Novel Serine Hydrolase, LipN, Implicated in the Virulence of Mycobacterium tuberculosis

Daniel Schemenauer Butler University
Faculty Sponsor(s): R. Jeremy Johnson Butler University
Tuberculosis (TB) remains one of the most prevalent diseases in the world, infecting one third of the world’s population. The causative agent, Mycobacterium tuberculosis, relies on a plethora of lipases to maintain an infection. With their central roles in infection, lipases have become a viable target for drug development. This research aimed to determine the substrate specificity, biochemical properties, and potential structural information of LipN, one proposed lipase. Initially, wild type LipN was expressed in E. coli, purified, and its substrate specificity characterized against 35 latent fluorophore substrates. LipN demonstrated the highest catalytic efficiency against small carbon chains with a kcat/Km over 10^5 M-1s-1 for its best single acetyl ester substrate and maintained high activity (kcat/Km > 10^4) with small polar groups. LipN’s preference for short nonpolar substrates suggests its physiological role as an esterase rather than a lipase. To categorize the structural factors controlling this substrate specificity, nine variants of LipN were made with substitutions in predicted binding pocket and active site residues and their relative kinetics against the top three ester substrates were characterized. Based on this analysis, the serine and histidine residues of the catalytic triad were positively identified, with 100-fold decreases in catalytic activity upon their substitution. The aspartate residue in the catalytic triad was tentatively assigned, as multiple aspartate variants showed similar reductions in catalytic efficiency. Future work will be aimed at assigning the catalytic triad as well obtaining a three-dimensional structure of LipN to confirm the structural features controlling its substrate specificity.
Biochemistry & Molecular Biology
Oral Presentation

When & Where

11:30 AM
Gallahue Hall 105