title: Characterization of the channel constriction allowing the access of the substrate to the active site of yeast oxidosqualene cyclase.
abstract: In oxidosqualene cyclases (OSCs), an enzyme which has been extensively studied as a target for hypocholesterolemic or antifungal drugs, a lipophilic channel connects the surface of the protein with the active site cavity. Active site and channel are separated by a narrow constriction operating as a mobile gate for the substrate passage. In Saccharomyces cerevisiae OSC, two aminoacidic residues of the channel/constriction apparatus, Ala525 and Glu526, were previously showed as critical for maintaining the enzyme functionality. In this work sixteen novel mutants, each bearing a substitution at or around the channel constrictions, were tested for their enzymatic activity. Modelling studies showed that the most functionality-lowering substitutions deeply alter the H-bond network involving the channel/constriction apparatus. A rotation of Tyr239 is proposed as part of the mechanism permitting the access of the substrate to the active site. The inhibition of OSC by squalene was used as a tool for understanding whether the residues under study are involved in a pre-catalytic selection and docking of the substrate oxidosqualene.
authors.affiliation: Dipartimento di Scienza e Tecnologia del Farmaco, Facoltà di Farmacia, Università degli Studi di Torino, Turin, Italy.
authors.forename: Simonetta
authors.initials: S
authors.lastname: Oliaro-Bosso
authors.forename: Giulia
authors.initials: G
authors.lastname: Caron
authors.forename: Silvia
authors.initials: S
authors.lastname: Taramino
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ermondi
authors.forename: Franca
authors.initials: F
authors.lastname: Viola
authors.forename: Gianni
authors.initials: G
authors.lastname: Balliano
mesh_terms: D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006860:Hydrogen Bonding / Q000187:drug effects; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D013185:Squalene / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D014170:Transformation, Genetic / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007473:Ion Channels; D050505:Mutant Proteins; D014443:Tyrosine; D013185:Squalene; D019751:Intramolecular Transferases; C021924:lanosterol synthase