Serine hydrolase profiling on Arabidopsis

Activity-based protein profiling (ABPP) is a powerful method to display enzyme activities in proteomes, and provides crucial information on enzyme activity, rather than protein or transcript abundance. We applied ABPP using fluorophosphonate (FP)-based probes to display the activities of serine (Ser) hydrolases in the model plant Arabidopsis thaliana. AARE; serine carboxypeptidase-like SNG1 and BRS1; carboxylesterase-like CXE12; methylesterases MES2 and MES3; and S-formylglutathione hydrolase SFGH, the majority of these serine hydrolases have not been described before. We studied transiently expressed SNG1 and investigated plants infected with the fungal pathogen Botrytis cinerea. Besides the down regulation of several Arabidopsis Ser hydrolase activities during Botrytis infection, we detected the activities of Botrytis-derived cutinases and lipases, which are thought to contribute to pathogenicity. Serine hydrolases comprise a large collection of enzymes from different structural classes that fulfill diverse biochemical roles such as proteases, lipases, esterases, and transferases. The Arabidopsis genome encodes for hundreds of serine hydrolases that belong to dozens of large multigene families (The Arabidopsis Genome Initiative, 2000).Common to these enzymes is that the active site contains an activated serine residue that performs the nucleophilic attack on the substrate, resulting in a covalent intermediate. To study the diverse roles of serine hydrolases in plants and in other organisms in detail, it is essential to display the activities of these enzymes because enzyme activities depend on various post-translational processes like phosphorylation, nitrosylation, processing, cofactors and inhibitors, and prediction of enzyme activities from transcriptomic or proteomic data can be misleading. Serine hydrolase activities can be displayed through activity-based protein profiling (ABPP). ABPP is based on the use of fluorescent or biotinylated small molecules that irreversibly react with the active site of enzymes in a mechanism-dependent manner, and has 2 Active site accessibility and reactivity is an important indication for enzyme activity (Kobe and Kemp, 1999). Labeled enzymes can be displayed on protein gels and blots or identified by mass spectrometry. A frequently used probe for serine hydrolases is based on fluorophosphonate (FP), which is also the reactive moiety in the broad-range serine hydrolase inhibitor DFP (diisopropyl fluorophosphonate). When used on mammalian extracts, FP displays dozens of serine hydrolase activities, including proteases, lipases and esterases (Liu et al., 1999; Kidd et al., 2001). FP probes do not label zymogen or inhibitor-bound serine hydrolases demonstrating that FP probes label only active enzymes in a proteome (Kidd et al., 2001). Serine hydrolase profiling with FP has proved extremely …