2001 Fiscal Year Final Research Report Summary
Structure-Function Analysis of A Novel Oxidosqualene Cyclase Involved in the Biosynthesis Antibiotics
| Project/Area Number |
12680597
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | Univ. Shizuoka |
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Principal Investigator |
ABE Ikuro Univ. Shizuoka, School Pharm. Sci., Lecturer, 薬学部, 講師 (40305496)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Oxidosqualene Cyclase / Steroidal Antibiotie / Lanosterol / Protosterol / Enzyme / Strucuture Function Relationship / Enzyme Purification / Cloning |
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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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