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.
Highlights
In sterol biosynthesis, oxidosqualene cyclases (OSCs) catalyze the most outstanding structural alteration step of the pathway: the shaping of the totally open triterpene intermediate 2,3-oxidosqualene, generated by the mevalonate pathway, into a polycyclic steroid, namely lanosterol in non-photosynthetic eukaryotes or cycloartenol in photosynthetic eukaryotes
Milestones of this long scientific pathway are the solution of the crystal structure of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius in 1997 [3] and human OSC (HsaOSC) in 2004 [4]
The structural analyses of both SHC and HsaOSC revealed the essential features of the active sites, and evidenced the presence of a lipophilic channel connecting the surface of the protein with the active site cavity
Summary
Oxidosqualene cyclases (OSCs) catalyze the most outstanding structural alteration step of the pathway: the shaping of the totally open triterpene intermediate 2,3-oxidosqualene, generated by the mevalonate pathway, into a polycyclic steroid, namely lanosterol in non-photosynthetic eukaryotes or cycloartenol in photosynthetic eukaryotes. Some of them were designed to extend the study of the previously selected position Ala525, others were suggested by the interactions shown in the modelling study by the residues His193, Asn211, His291 [6], others, were designed on the basis of the structural role assigned to the corresponding residues Tyr237 and Cys233 in HsaOSC [4] To make it possible to compare results of the present and previous study, all the novel mutants have been built starting from the mutant C457D, housing a CRD mutation in the active site cavity, used in our first sitedirected mutagenesis study [6]
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