Molecular cloning, expression, and site-directed mutations of oxidosqualene cyclase from Cephalosporium caerulens.
A cDNA for oxidosqualene:lanosterol cyclase (OSLC) was cloned and sequenced from the fungus Cephalosporium caerulens, that produces a steroidal antibiotic, helvolic acid. A 2280 bp open reading frame encoded an M(r) 87078 protein with 760 amino acids. The cDNA was functionally expressed in the OSLC-deficient mutant GIL77 strain of Saccharomyces cerevisiae. A truncated recombinant enzyme (Delta49N) 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 for 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.
Abe
I
I
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, 422-8526, Shizuoka, Japan. abei@ys7.u-shizuoka-ken.ac.jp
Naito
K
K
Takagi
Y
Y
Noguchi
H
H
D000164:Acremonium / Q000201:enzymology / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D005672:Fusidic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D005822:Genetic Vectors; D019751:Intramolecular Transferases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D017510:Protein Folding; D016415:Sequence Alignment
D005672:Fusidic Acid; D019751:Intramolecular Transferases; C021924:lanosterol synthase; C011267:helvolic acid