Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure-activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates. Two structurally homologous bacterial esterases were screened against this library, refining their previously broad overlapping substrate specificity. esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr-119 in ybfF and His-187 in Rv0045c; reciprocal substitution of these residues shifted each esterase's substrate preference. This work demonstrates that the selectivity of esterases is tuned based on transition state stabilization, identifies thioethers as an underutilized functional group for esterase substrates, and provides a rapid method for differentiating structural isozymes. This SAR library could have multifaceted future applications, including imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.

Ubiquitous cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. These diverse substrates and functions however complicate a priori prediction of their substrate preferences and biological functions. Analogous to substrate activity screening for serine protease activity, we designed a 36‐member structure activity relationship (SAR) library of fluorogenic esterase substrates with low background hydrolysis, high sensitivity, and modular, straightforward synthesis. In three parallel substrate series containing alkyl, ether, and thioether substituents, the SAR library systemically interrogates esterase preference for chain length, branching patterns, polarity, and hydrogen bonding to differentiate common classes of esterase substrates. Applying this library against two structurally homologous bacterial esterases, previously broad overlapping substrate specificity was refined to preferences for γ‐position thioethers and ethers for ybfF from Vibrio cholerae and branched substrates with and without thioethers for Rv0045c from Mycobacterium tuberculosis. Structural control over this substrate differentiation was then assigned to individual substrate selectivity residues of Tyr116 in ybfF and His187 in Rv0045c whose reciprocal substitution inverted each esterase's substrate preference. This SAR esterase library could have multi‐faceted future applications including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health (NIH 1 R15 GM110641‐01A1).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.