MOLECULAR CLONING, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF RAT OXIDOSQUALENE CYCLASE CDNA

摘要

cDNA encoding rat oxidosqualene lanosterol-cyclase [Lanosterol synthase; (S) -2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization. An open reading frame of 2199 bp encodes a M(r)83,321 protein with 733 amino acids. The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-). Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif, The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs. The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide. Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain. A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae. The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor [H-3] 29-MOS. [References: 50]