Building a Synthetic Library of Fluorogenic Ester Substrates to Analyze Serine Hydrolases

Andrew Koelper Butler University
Faculty Sponsor(s): Geoffrey Hoops Butler University
Mycobacterium tuberculosis, the organism responsible for the respiratory infection tuberculosis, has evolved multi-drug resistance strains, introducing the need for novel drug targets. Serine hydrolases with diverse roles in lipid metabolism and dormant growth have been proposed as novel targets for tuberculosis. Although many serine hydrolases from TB have been identified by proteomics and activity based protein profiling, the natural substrates and biological reactions for these serine hydrolases are still unknown. To globally investigate the natural substrates for mycobacterial serine hydrolases, we synthesized a structure activity library of 32 fluorogenic ester substrates to screen against mycobacterial hydrolases. The library contains acyloxy methyl ester derivatives of fluorescein with small variations in ester functionalities to mimic mycobacterial lipids. To confirm the utility of the fluorogenic library for characterizing serine hydrolases, two structurally homologous bacterial serine hydrolases, Rv0045c from M. tuberculosis and ybfF from V. cholera, were screened against the library of fluorogenic substrates to determine the catalytic efficiency of these enzymes on each substrate. Rv0045c and ybfF both favor short, 3-atom, ether chains with the heteroatom in the β−position. In addition, comparative kinetic analysis of hydrolase variants with substitutions in their binding pocket provided insight into the substrate specificity of the enzymes, and thereby the natural substrate. Catalytic efficiency deviations from the WT, could be a result of steric hindrance, hydrogen bond interactions, and localized acid-base chemistry. Future work would be aimed at making more variants and determining the structural features, which contribute to their substrate specificity.
Biochemistry & Molecular Biology
Poster Presentation

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Irwin Library 3rd Floor