title: A Cutinase from Trichoderma reesei with a lid-covered active site and kinetic properties of true lipases.
abstract: Cutinases belong to the α/β-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded β-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as β-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Aix Marseille Université, 13284 Marseille Cedex 09, France; Architecture et Fonction des Macromolécules Biologiques, UMR7257, Centre National de la Recherche Scientifique, 13288 Marseille Cedex 09, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Roussel
authors.affiliation: Aix Marseille Université, UMR7282, Centre National de la Recherche Scientifique, 13402 Marseille Cedex 20, France.
authors.forename: Sawsan
authors.initials: S
authors.lastname: Amara
authors.affiliation: VTT Biotechnology, P. O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Nyyssölä
authors.affiliation: Aix Marseille Université, UMR7282, Centre National de la Recherche Scientifique, 13402 Marseille Cedex 20, France.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Mateos-Diaz
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Aix Marseille Université, 13284 Marseille Cedex 09, France; Architecture et Fonction des Macromolécules Biologiques, UMR7257, Centre National de la Recherche Scientifique, 13288 Marseille Cedex 09, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Blangy
authors.affiliation: VTT Biotechnology, P. O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Hanna
authors.initials: H
authors.lastname: Kontkanen
authors.affiliation: VTT Biotechnology, P. O. Box 1000, FIN-02044 VTT, Finland.
authors.forename: Ann
authors.initials: A
authors.lastname: Westerholm-Parvinen
authors.affiliation: Aix Marseille Université, UMR7282, Centre National de la Recherche Scientifique, 13402 Marseille Cedex 20, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Carrière
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Aix Marseille Université, 13284 Marseille Cedex 09, France; Architecture et Fonction des Macromolécules Biologiques, UMR7257, Centre National de la Recherche Scientifique, 13288 Marseille Cedex 09, France. Electronic address: cambillau@afmb.univ-mrs.fr.
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008066:Lipolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D063065:Organophosphonates / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D063065:Organophosphonates; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D008049:Lipase
keywords: Trichoderma reesei; X-ray structure; cutinase; interfacial activation; lipase