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.

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell-specific quantification of protein abundance changes, we sought to apply an enzyme-activated cellular fluorescence system in vivo by delivering ester-masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES-1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.

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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.