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.

A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin

“…Possibly, the substrate specificity of the L. clavatum a-onocerin synthases could rely on amino acids in the OSC substrate channel. In fact, a-onocerin synthases of O. spinosa differ from those in L. clavatum in amino acids that correspond to residues described in the OSC substrate channel, in particular HsLAS Cys-233 and Ile-524, which are both involved in the passage of the substrate to the active site (Thoma et al, 2004), His-289 is involved in the hydrogen bond network of the channel constriction loop (Oliaro-Bosso et al, 2005), and Tyr-237 has been proposed to allow access of the substrate to the catalytic cavity by rotating (Oliaro-Bosso et al, 2011). Lycopods arose early on in the plant phylogeny; hence, LcLCC and LcLCD have perhaps had more time for substrate specialization.…”

Engineering Saccharomyces cerevisiae for Hyperproduction of β-Amyrin by Mitigating the Inhibition Effect of Squalene on β-Amyrin Synthase

“…cerevisiae. Certainly, the activity of ERG7 might also be inhibited by squalene, whereas the strong competition of the ergosterol synthesis pathway implied that the activity of ERG7 was higher than that of GgbAS1 …”

“…Certainly, the activity of ERG7 might also be inhibited by squalene, whereas the strong competition of the ergosterol synthesis pathway implied that the activity of ERG7 was higher than that of GgbAS1. 16 Peroxisome Compartmentalization for β-Amyrin Production. In S. cerevisiae, GgbAS1 and the synthesis of squalene were both localized in the cytosol and likely in the endoplasmic reticulum and lipid droplets.…”

“…It was reported that the activity of lanosterol synthases, which belonged to OSCs, was inhibited by squalene in S. cerevisiae. , As a member of OSCs, the activity of bAS1 was also inhibited by squalene analogues in plant cell suspension cultures . Therefore, it was speculated that the activity of bAS1 might be inhibited by squalene in S.…”

“…14 It was reported that the activity of lanosterol synthases, which belonged to OSCs, was inhibited by squalene in S. cerevisiae. 15,16 As a member of OSCs, the activity of bAS1 was also inhibited by squalene analogues in plant cell suspension cultures. 17 pathway were localized in the cytosol, suggesting that subcellular compartmentalization might be a good strategy to avoid the inhibition effect of squalene accumulation on the activity of bAS1.…”