Δ7-Sterol-C5-desaturase:: molecular characterization and functional expression of wild-type and mutant alleles

An Arabidopsis thaliana recessive monogenic mutant (stel-1) presenting a deficiency of the Delta(7)-sterol-C5(6)-desaturase step in the sterol pathway has been reported previously [12]. To further characterize stel-1, Arabidopsis, Nicotiana tabacum and Homo sapiens cDNAs encoding Delta 7-sterol-C5(6)-desaturases were isolated and identified on the basis of their ability to restore ergosterol synthesis in erg3, a yeast null mutant whose gene encoding the Delta 7-sterol-C5(6)-desaturase was disrupted. Overexpression of the Arabidopsis cDNA driven by a 35S promoter in transgenic stel-1 plants led to full complementation of the mutant. This result demonstrates that STE1 was the impaired component in the desaturation system. Four independent reverse transcriptions of stel-1 RNA followed by polymerase chain reactions (RT-PCRs), yielded a single product. Alignment of the wild-type ORF with the RT-PCR derived stel-1 ORF revealed a single amino acid substitution: Thr-114 in the wild-type is changed to lie in stel-1. Expression in erg3 resulted in a 6-fold lowered efficiency of the stel-1 ORF in complementing the yeast biosynthetic pathway when compared to the wild-type ORE The presence of this mutation in the mutant stel-1 genomic sequence land no additional modification between stel-1 and wild-type genes) demonstrates that the change of the Thr-114 to Ile is necessary and sufficient to create the leaky allele stel-1. The occurrence of a hydroxylated amino acid (Thr or Ser) at the position corresponding to Thr-114 in the five Delta(7)-sterol-C5(6) desaturases identified so far suggests that this amino acid is important for normal enzymatic function. Nomenclature: erg3, a Saccharomyces cerevisiae Delta(7)-sterol-C5(6)-desaturase mutant [2]; stel-1, an Arabidopsis Delta 7-sterol-C5(6)-desaturase mutant; ERG3, the protein encoded by ERG3, the wild-type yeast Delta(7)-sterol-C5(6)-desaturase gene; STE1, the protein encoded by STE1, the wild-type Arabidopsis Delta(7)-sterol-C5(6)-desaturase gene. Sterol nomenclature: lanosterol, 4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol; cycloartenol, 4,4,14 alpha-trimethyl-9 beta, 19-cyclo-5 alpha-cholest-24-en-3 beta-ol; episterol, 5 alpha-ergost-7,24(24(1))-dien-3 beta-ol; Delta(7)-avenasterol, 5 alpha-stigmast-7,Z-24(24(1))-dien-3 beta-ol; 5-dehydroepisterol, ergosta-5,7,24(24(1))-dien-3 beta-ol; 5-dehydro-Delta(7)-avenasterol, stigmasta-5,7,2-24(24(1))-dien-3 beta-ol; ergosterol, ergosta-5,7,E-22-dien-3 beta-ol; campesterol, 24R-24-methyl-cholest-5-en-3 beta-ol; 22-dihydrobrassicasterol, 24S-24-methyl-cholest-5-en-3 beta-ol; sitosterol, 24R-24-ethyl-cholest-5-en-3 beta-ol; stigmasterol, 24S-24-ethyl-cholesta-5,E-22-dien-3 beta-ol.