A Cutinase from Trichoderma reesei with a Lid-covered Active Site and Kinetic Properties of True Lipases defines a New Class of Lipolytic Enzymes
Mateos-Diaz E.1, Amara S.1, Roussel A.2, Nyyssölä A.3, Blangy S.2, Kontkanen H.3, Westerholm-Parvinen A.3, Cambillau C. 2, Carrière F1,
1 Centre National de la Recherche Scientifique, Aix-Marseille Université, UMR7282 Enzymology at Interfaces and Physiology of Lipolysis, Marseille
2 Centre National de la Recherche Scientifique, Aix-Marseille Université, UMR 7257 Architecture et Fonction des Macromolécules Biologiques, Marseille, France.
3 VTT Biotechnology, VTT, Finland.

Cutinases belong to the a/b hydrolase fold family of enzymes and degrade cutin as well as various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated as well as 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 -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 b-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.