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Screening for novel long‐chain bacterial esterase activity
Author(s) -
Jozwiakowski Jakob Leland,
Johnson R. Jeremy
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.796.10
Subject(s) - hydrolase , esterase , biochemistry , catalytic triad , chemistry , enzyme , escherichia coli , amidase , thioesterase , active site , hydrolysis , biosynthesis , gene
Hydrolases are ubiquitous cellular enzymes with more than 1200 different hydrolases, >1/3 of all enzyme structures in the PDB, and 13 different enzyme commission subclasses. Hydrolases are also fairly promiscuous and can also catalyze hydrolysis reactions on varied substrates including esters, amides, thioesters, phosphoric acid esters, and acid anhydrides. All of these reactions originate from the same α/β hydrolase protein fold and utilize the catalytic triad, suggesting great plasticity and generality in this reaction. Using a fluorogenic mimic of palmitic acid linked molecules, we performed a high‐throughput screen in Escherichia coli searching for previously unidentified depalmitoylase activity. Simultaneous analysis of forty‐five different hydrolase knockout strains with this fluorogenic probe identified a single uncharacterized esterase with high specificity for the probe. To confirm this reactivity, this hydrolase was heterologously expressed, purified to homogeneity, and characterized for its detailed substrate specificity using a library of fluorogenic esters. Our screen highlighted the potential novel hydrolase reactivity present within even model organisms like E. coli that could have applications in protein engineering or biological imaging. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .