Author:
Font-Farre Maria,Brown Daniel,Toth Reka,Mahadevan Chidambareswaren,Brazier-Hicks Melissa,Morimoto Kyoko,Kaschani Farnusch,Sinclair John,Dale Richard,Hall Samantha,Morris Melloney,Kaiser Markus,Wright Aaron T.,Burton Jonathan,van der Hoorn Renier A. L.
Abstract
AbstractEukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.
Funder
Biotechnology and Biological Sciences Research Council
European Research Council
Deutsche Forschungsgemeinschaft
Publisher
Springer Science and Business Media LLC