Abstract
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. Vibrio cholerae esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from Mycobacterium tuberculosis 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 in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.
Highlights
The authors declare that they have no conflicts of interest with the contents of this article
We previously developed a small, general library of fluorogenic ester substrates based on acyloxymethyl ether fluorescein
We propose that our library design could have multifaceted future applications, including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design
Summary
We applied this library to broadly characterize the structural factors controlling the substrate specificity of esterases, to propose biological functions for uncharacterized esterases, and to identify unusual biocatalytic reactions [31, 33, 36, 41,42,43] This preliminary fluorogenic library provided sensitive detection of even weak binding substrates within a high-throughput and straightforward assay design [35, 41,42,43]. We describe the development and application of a refined SAR fluorogenic library for pinpointing the substrate specificity profile of esterases We apply this library to differentiating the substrate specificity profiles of two structural isozymes with high structural similarity. We propose that our library design could have multifaceted future applications, including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design
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