Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen.
Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.
Corey
E J
EJ
Department of Chemistry, Harvard University, Cambridge, MA 02138.
Matsuda
S P
SP
Bartel
B
B
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D019751:Intramolecular Transferases; D007535:Isomerases / Q000096:biosynthesis* / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010840:Phytosterols / Q000096:biosynthesis / Q000737:chemistry; D010957:Plasmids; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D016339:Spectrometry, Mass, Fast Atom Bombardment; D017550:Spectroscopy, Fourier Transform Infrared; D014315:Triterpenes
D010840:Phytosterols; D014315:Triterpenes; D012313:RNA; D004247:DNA; D007535:Isomerases; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase; C021924:lanosterol synthase; C021923:cycloartenol synthase; C100089:cycloartenol