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Abstract Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, perhaps best known for inactivating N -acylethanolamine lipid mediators. However, FAAH enzymes hydrolyze a wide range of acylamide substrates. Analysis of FAAHs from multiple angiosperm species revealed two conserved phylogenetic groups that differed in key conserved residues in the substrate binding pocket. While the foundation group of plant FAAHs, designated FAAH1, has been studied at the structural and functional level in Arabidopsis thaliana , nothing is known about FAAH2 members. Here, we combined computational and biochemical approaches to compare the structural and enzymatic properties of two FAAH isoforms in the legume Medicago truncatula designated MtFAAH1 and MtFAAH2a. Differences in structural and physicochemical properties of the substrate binding pockets, predicted from homology modeling, molecular docking, and molecular dynamic simulation experiments, suggested that these two FAAH isoforms would exhibit differences in their amidohydrolase activity profiles. Indeed, kinetic studies of purified, recombinant MtFAAHs indicated a reciprocal preference for acylamide substrates with MtFAAH1 more efficiently utilizing long-chain acylamides, and MtFAAH2a more efficiently hydrolyzing short-chain and aromatic acylamides. This first report of the enzymatic behavior of two phylogenetically distinct plant FAAHs will provide a foundation for further investigations regarding FAAH isoforms in legumes and other plant species.
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Ex vivo lipidomics reveal monoacylglycerols as substrates for a fatty acid amide hydrolase in the legume Medicago truncatula
Fatty acid amide hydrolase (FAAH) is a conserved hydrolase in eukaryotes with promiscuous activity toward a range of acylamide substrates. The native substrate repertoire for FAAH has just begun to be explored in plant systems outside the modelArabidopsis thaliana. Here, we usedex vivolipidomics to identify potential endogenous substrates forMedicago truncatulaFAAH1 (MtFAAH1). We incubated recombinant MtFAAH1 with lipid mixtures extracted fromM. truncatulaand resolved their profiles via gas chromatography–mass spectrometry (GC–MS). Data revealed that besidesN‐acylethanolamines (NAEs),sn‐1orsn‐2isomers of monoacylglycerols (MAGs) were substrates for MtFAAH1. Combined within vitroand computational approaches, our data support both amidase and esterase activities for MtFAAH1. MAG‐mediated hydrolysis via MtFAAH1 may be linked to biological roles that are yet to be discovered.
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- Award ID(s):
- 2051636
- PAR ID:
- 10580523
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- FEBS Letters
- Volume:
- 598
- Issue:
- 15
- ISSN:
- 0014-5793
- Format(s):
- Medium: X Size: p. 1839-1854
- Size(s):
- p. 1839-1854
- Sponsoring Org:
- National Science Foundation
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