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Showing papers on "Lanosterol synthase published in 2009"


Journal ArticleDOI
TL;DR: There is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells, and the pathway is designated “the Lanosterol pathway.”
Abstract: The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-13C2H3]mevalonate with Arabidopsis seedlings. Applying 13C-{1H}{2H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol “the lanosterol pathway.” LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as β-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites.

187 citations


Journal ArticleDOI
TL;DR: LC-MS and NMR analyses of the accumulated product in the host cells showed that the product of At1 g78950 is beta-amyrin, indicating that At1g78950 encodes a beta-Amyrin synthase.

57 citations


Journal ArticleDOI
TL;DR: Evidence is emerging that endogenous 24(S),25-epoxycholesterol can work at several levels to control acute cholesterol homeostasis, and this oxysterol appears to serve as a measure of cholesterol synthesis and to protect against surges in the production of this potentially cytotoxic molecule.

38 citations


Journal ArticleDOI
TL;DR: The data indicate that cyclase inhibitors are capable of producing CYP3A4 and CYP2B6 induction in primary cultured human hepatocytes, and that an endogenous squalene metabolite is a conserved intracrine activator of PXR.
Abstract: The effects of [4′-(6-allyl-methyl-amino-hexyloxy)-2′-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071), an inhibitor of 2,3-oxidosqualene:lanosterol cyclase (cyclase), were evaluated on CYP3A4 and CYP2B6 mRNA content in primary cultured human hepatocytes. In seven hepatocyte culture preparations, 24-h treatment with 3, 10, or 30 μM Ro 48-8071 produced median increases in CYP3A4 mRNA content that were 2.2-, 7.1-, and 8.5-fold greater than untreated control, respectively, and produced increases in CYP2B6 mRNA content that were 3.0-, 4.6-, and 3.4-fold greater than control, respectively. Increases in CYP3A4 immunoreactive protein content were also measured in Ro 48-8071-treated hepatocytes. To evaluate the effects of cyclase inhibitor treatments further, a pregnane X receptor (PXR)-responsive transactivation assay in HepG2 cells was used. Ro 48-8071, trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine (BIBX 79), and 3β-(2-diethylaminoethoxy)androst-5-en-17-one HCl (U18666A) induced luciferase expression from a PXR-responsive reporter with EC50s of 0.113, 0.916, and 0.294 μM, respectively. Treatment of the HepG2 system with (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3′-bithiophen-5-yl)methoxy]benzenemethanamine (NB-598), an inhibitor of squalene monooxygenase, at concentrations sufficient to achieve cholesterol biosynthesis inhibition significantly inhibited cyclase inhibitor-mediated, but not rifampicin-mediated, reporter induction. Direct treatment of the HepG2 system with 1 to 10 μM squalene 2,3:22,23-dioxide, but not squalene 2,3-oxide, significantly activated PXR-responsive reporter expression. Also, squalene 2,3:22,23-dioxide bound to human PXR in vitro with an IC50 of 3.35 μM. These data indicate that cyclase inhibitors are capable of producing CYP3A4 and CYP2B6 induction in primary cultured human hepatocytes, and that an endogenous squalene metabolite is a conserved intracrine activator of PXR.

14 citations


Journal ArticleDOI
TL;DR: The function of ERG7Y99 residue from Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) was analyzed by constructing deletion and site-saturated mutants as discussed by the authors.

14 citations


Journal ArticleDOI
TL;DR: In cell cultures of both human keratinocytes and mouse 3T3 fibroblasts, the observed inhibition of cholesterol biosynthesis was selective for oxidosqualene cyclase.
Abstract: Human and murine lanosterol synthases (EC 5.4.99.7) were studied as targets of a series of umbelliferone aminoalkyl derivatives previously tested as inhibitors of oxidosqualene cyclases from other eukaryotes. Tests were carried out on cell cultures of human keratinocytes and mouse 3T3 fibroblasts incubated with radiolabeled acetate, and on homogenates prepared from yeast cells expressing human lanosterol synthase, incubated with radiolabeled oxidosqualene. In cell cultures of both human keratinocytes and mouse 3T3 fibroblasts, the observed inhibition of cholesterol biosynthesis was selective for oxidosqualene cyclase. The most active compounds bear an allylmethylamino chain in position-7 of the coumarin ring. The inhibition was critically dependent on the position and length of the inhibitor side chain, as well as on the type of aminoalkyl group inserted at the end of the same chain. Molecular docking analyses, carried out to clarify details of inhibitors/enzyme interactions, proved useful to explain the ...

13 citations


Dissertation
01 Jan 2009
TL;DR: Functional characterization of three oxidosqualene cyclase genes from the model plant Arabidopsis thaliana that encode enzymes with novel catalytic functions are described, establishing that plant lanosterol synthases comprise a catalytically distinct class of lanosterl synthases.
Abstract: This thesis describes functional characterization of three oxidosqualene cyclase genes (At1g78955, At3g45130, and At4g15340 ) from the model plant Arabidopsis thaliana that encode enzymes with novel catalytic functions. Oxidosqualene cyclases are a family of membrane proteins that convert the acyclic substrate oxidosqualene into polycyclic products with many chiral centers. The complex mechanistic pathways and relevant catalytic motifs can be elucidated through judicious applications of mutagenesis, heterologous expression in combination with a genome mining approach, and protein modeling. Functional characterization of oxidosqualene cyclases allows improved understanding on how these proteins guide catalytic reactions and how protein-substrate interactions affect the reaction outcome, as well as identification of triterpenes with novel structures and stereochemistry. This work describes characterization of Arabidopsis oxidosqualene cyclases, including the first plant lanosterol synthase (LSS1), an enzyme with novel catalytic motifs different from those previously observed in animal, fungal, and trypanosomal lanosterol synthases, establishing that plant lanosterol synthases comprise a catalytically distinct class of lanosterol synthases. Phylogenetic analysis reveals that lanosterol synthases are broadly distributed in eudicots but evolved independently from those in animals and fungi. Discovery of plant lanosterol synthase also suggests lanosterol as precursor for plant 4,4-dimethyl-Delta8 sterols. Additional mutagenesis experiments on Arabidopsis lanosterol synthase (Asn477His and Va1481Ile) allowed for introduction of cycloartenol activity in a lanosterol synthase background, providing the best example of engineered biosynthesis of cyclopropyl structures known to date. This thesis also describes the first enzyme (camelliol C synthase, CAMS1) that efficiently blocks B-ring formation to make a monocyclic triterpene camelliol C. Phylogenetic analysis reveals that this cyclase has evolved from enzymes that generate pentacycles, and sequence comparison between oxidosqualene cyclases with different catalytic functions allowed for identification of key residues that increases steric bulk in the active site to promote monocycle formation. Finally, this thesis describes an enzyme arabidiol synthase (PEN1) that produces the tricyclic triterpene diol arabidiol. Analysis of the arabidiol structure and characterization of numerous minor products of arabidiol synthase, including several novel compounds, resulted in formulation of a general rule for water addition in triterpene biosynthesis and an explanation for the domination of deprotonation over water addition in triterpene biosynthesis.

2 citations