Premium
Global substrate specificity of mycobacterial serine hydrolases
Author(s) -
Johnson R. Jeremy,
Bassett Braden,
Waibel Brent,
White Alex,
Hansen Heather,
Stephens Dominique,
Koelper Andrew,
Hoops Geoffrey
Publication year - 2017
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.31.1_supplement.767.11
Subject(s) - serine hydrolase , serine , hydrolase , mycobacterium tuberculosis , mycobacterium , biochemistry , enzyme , mycobacterium smegmatis , chemistry , biology , microbiology and biotechnology , bacteria , tuberculosis , medicine , pathology , genetics
One third of the world's population is estimated to be infected with a dormant TB infection. M. tuberculosis survives within this dormant state by engulfing host cell lipid bodies and then accumulating significant intracellular stores of lipids. Among the proteins required for lipid metabolism in M. tuberculosis are a significant number of uncharacterized enzymes predicted to encode serine hydrolases and specifically lipases. Using a streamlined synthetic method, a library of hydrolase substrates based on acyloxy methyl ether fluorescein were expanded to better represent the natural lipodomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global substrate specificity of mycobacterial serine hydrolases under normal and dormant growth conditions. Conformation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors. The hydrolases responsible for the global substrate specificity of one highly active fluorogenic substrate were then isolated using a combination of native protein gel‐resolved activity measurements and mass spectrometry identification. Substrates that were highly activated in Mycobacterium were also tested in other common bacterial strains to determine the orthogonality of the mycobacterial hydrolase reactions. Using the highly activated fluorogenic substrates, future work will compare the serine hydrolase profiles between different strains of infectious Mycobacterium to aid in diagnostics and drug discovery. Support or Funding Information Funded by NIH 1R15GM110641‐01A1