Abstract 1511 Screening for serine hydrolase activity in E. coli against fluorogenic ester substrates

Antibiotic resistant bacteria are constantly evolving and pose a threat to public health. One method to combat antibiotic resistance is to repurpose or redirect nonspecific drugs through their modification and targeting as prodrugs. Prodrug development requires an understanding of the activity and substrate specificity of a target's entire enzyme repertoire. One class of enzymes used extensively in human drug targeting but with limited current antibiotic applications are serine hydrolases, a broad enzyme superfamily central to catalyzing essential biochemical reactions. Serine hydrolases constitute over 1% of the genes in Escherichia coli, making it a bacterium of interest in prodrug research. My research objective was to use in-gel enzyme imaging based on native PAGE separation of E. coli whole cell lysates to analyze the substrate specificity of serine hydrolases in E. coli against 23 fluorogenic ester substrates. Using fluorogenic esters combined with in-gel enzyme analysis allowed us to simultaneously screen each of these 23 substrates against multiple E. coli serine hydrolases and to identify unique substrate reactivity patterns present in E. coli. Among ester substrates, phenylthioacetate ester, an alkyl thioether substituent, allowed for the visualization of two unique enzyme bands. This ester substrate was then combined with fifty-six E. coli knockout strains to trace this measured E. coli activity to two esterases – yjfP and tesA. Future work will revolve around characterizing the shifting serine hydrolase activity of E. coli across variable growth conditions with the potential of using unique ester reactivity in antibiotic prodrug development.