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Fluorogenic structure activity library pinpoints molecular variations in the substrate specificity of structurally homologous esterases
Author(s) -
Johnson R. Jeremy,
White Alex,
Koelper Andrew,
Russell Arielle,
Larsen Erik,
Hoops Geoffrey
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.530.7
Subject(s) - esterase , substrate (aquarium) , chemistry , serine hydrolase , active site , stereochemistry , biochemistry , combinatorial chemistry , serine , biology , enzyme , ecology
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 Information This 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 .