title: Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets.
abstract: The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the "membrane-access" and the "acyl chain-binding" pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH's mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.
authors.affiliation: Laboratory of Molecular Modeling and Drug Discovery, Istituto Italiano di Tecnologia, Genova, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Palermo
authors.affiliation: CompuNet, Istituto Italiano di Tecnologia, Genova, Italy.
authors.forename: Inga
authors.initials: I
authors.lastname: Bauer
authors.affiliation: Laboratory of Computational Chemistry and Biochemistry, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Pablo
authors.initials: P
authors.lastname: Campomanes
authors.affiliation: CompuNet, Istituto Italiano di Tecnologia, Genova, Italy; Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cavalli
authors.affiliation: D3-PharmaChemistry, Istituto Italiano di Tecnologia, Genova, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Armirotti
authors.affiliation: CompuNet, Istituto Italiano di Tecnologia, Genova, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Girotto
authors.affiliation: Laboratory of Computational Chemistry and Biochemistry, Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Ursula
authors.initials: U
authors.lastname: Rothlisberger
authors.affiliation: Laboratory of Molecular Modeling and Drug Discovery, Istituto Italiano di Tecnologia, Genova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: De Vivo
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001095:Arachidonic Acids / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D019295:Computational Biology; D063388:Endocannabinoids / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation; D053284:Polyunsaturated Alkamides / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide