| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
A cDNA for oxidosqualene cyclase (OSC) was cloned and sequenced from the fungus Cephalosporium caerulens, that produces a steroidal antibiotic, helvolic acid. A 2,280-bp open reading frame encoded a Mr 87,078 protein with 760 amino acids, whose sequence showed ca. 37% identity with those of OSCs fromrat and yeast. The cDNA was functionally expressed in the OSLC-deficient mutant GIL77 strain of Saccharomyces cerevisiae. The recombinant enzyme afforded lanosterol as a singlele product. A truncated recombinant enzyme (Δ49N) starting from the second methionine (M50) residue was completely inactive, suggesting that ca. 30 additional hydrophilic amino acid residues at the N-terminal are essential fox the folding of the enzyme. Furthermore,, the active site residues, H234 and D456 (numbering in S. cerevisiae OSLC), were chosen for site-directed mutagenesis experiments; H234E, H234Y, H234F, D456E, D456N, and D456H mutants were inactive, while H234W and H234K mutants retained lanosterol forming activity. Purification of native protosterol synthase from the fungi is now in progress.
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