Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates

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...

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Bibliographic Details
Published in:ACS infectious diseases 2018-06, Vol.4 (6), p.904-911
Main Authors: Bassett, Braden, Waibel, Brent, White, Alex, Hansen, Heather, Stephens, Dominique, Koelper, Andrew, Larsen, Erik M., Kim, Charles, Glanzer, Adam, Lavis, Luke D., Hoops, Geoffrey C., Johnson, R. Jeremy
Format: Article
Language:English
Subjects:
Cluster Analysis
Esters
Fluorescent Dyes - chemistry
Fluorescent Dyes - metabolism
Hydrolysis
Molecular Structure
Mycobacterium - enzymology
Serine Endopeptidases - chemistry
Serine Endopeptidases - metabolism
Structure-Activity Relationship
Substrate Specificity
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Summary: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.
ISSN:2373-8227
2373-8227
DOI:10.1021/acsinfecdis.7b00263