Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA

Abstract

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

Sterol Metabolism

“…This result provides evidence that the Alicyclobacillus acidocaldarius and Zymomonas mobilis hopene synthases (Wendt et al, 1997), RNLAN, SCLAN and TBLAN : Rattus norvegicus (Abe and Prestwich, 1995), Saccharomyces cerevisiae (Shi et al, 1994) and Trypanosoma cruzi (Joubert et al, 2001) lanosterol synthases. ATCYC, DDCYC, PSCYC, GGCYC, PGCYC, LCCYC : Arabidopsis thaliana (Corey et al, 1993), Dictyostelium discoidum (Godzina et al, 2000), Pisum sativum, Glycyrrhiza glabra, Panax ginseng (Kushiro et al, 1998), Luffa cylindrica cycloartenol synthases . ATLUP1 to 5 : Arabidopsis thaliana triterpene synthases which cyclize 2,3-oxidosqualene into various pentacyclic triterpenes (Herrera et al, 1998 ;Husselstein-Muller et al, 2001 ;Kushiro et al, 2000).…”

“…2). Photosynthetic eukaryotes and certain non photosynthetic protists such as Acantamoeba polyphaga (Raederstorff and Rohmer, 1987) or Dictyostelium discoidum (Godzina et al, 2000;Nes et al, 1990) use CPI (EC 5.5.1.9) to convert pentacyclic cyclopropyl sterols to tetracyclic end-pathway sterols. Arabidopsis CPI was cloned by functional complementation of a S.cerevisiae mutant (Lovato et al, 2000).…”

Lewis Acid Catalyzed Benzylic Bromination

“… Representative cycloartenol synthases from Dictyostelium discoideum14 (DdiCAS1) and A. thaliana ( Ath CAS1), with representative lanosterol synthases from the fungi Saccharomyces cerevisiae15 (SceERG7) and Schizosaccharomyces pombe16 (SpoERG7) and the animals Homo sapiens17 (HsaERG7) and Rattus norvegicus18 (RnoERG7). Tyr410 (⧫), His477 (*), and Ile481 (▾) are labeled above the sequences.…”

Phytosterol Profiles, Genomes and Enzymes – An Overview

“…The growing access to genome mining is now rapidly broadening the understanding of the origin and distribution of sterol biosynthetic capabilities in the tree of life. The best example when considering sterol pathways is the widespread occurrence of cycloartenol (and cycloartenol synthase, CAS) in prokaryotes ( Bode et al, 2003 ) and eukaryotes like Dictyostelium discoidum ( Godzina et al, 2000 ; Meyer et al, 2002 ) or Naegleria ( Raederstorff and Rohmer, 1987 ). In fact, novel and massive genome and metagenome analysis coupled with ad hoc functional validation assays will facilitate the access to yet undisclosed sterol biosynthetic enzymes or more generally new pathways and therefore new aspects of cellular homeostasis.…”