Reaction catalyzed by lanosterol synthase is one of the most complex biosynthetic reactions found in nature, and its mechanism has been the subject of much research over three decades. Lanosterol synthase has attracted considerable interest from the biological and pharmaceutical viewpoints since it is situated in the center of sterol biosynthesis of eukaryotes. Membrane associated rat liver lanosterol synthase has been solubilized with Triton X-100 and for the first time purified to homogeneous protein in three steps (hydroxylapatide, isoelectric focussing and Mono Q). The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 75kD. Eleven independent amino acid sequences (Peptide-1 to -11) of the endogenous polypeptide were obtained by sequencing the internal peptides after Lys-C or lysylendopeptidase digestion of the purified enzyme. With all the available amino acid sequence information, we tried to clone cDNA of this enzyme by PCRs. After several trials, one with a pair of sense primer based on Peptide-8 and anti-sense primer based on Peptide-9 gave an amplified ca. 200-bp DNA fragment. This fragment was subcloned into pT7Blue(R) and sequenced. Based on the sequence of this fragment, the 5'- and 3'-ends of nucleotide sequences were elucidated by RACE strategy. The full nucleotide sequence revealed the presence of 2,199-bp ORF that encodes a 733 amino acid polypeptide with a molecular mass of 83,301Da and all of the sequences of eleven internal peptides are included. The PCR-amplified ORF has been inserted into pYES2, an expression vector in yeast, under the control of galactose-inducible promoter. Significant lanosterol synthase activity has been found in the homogenate of the transformed yeast, confirming its identity as lanosterol synthase cDNA. The deduced amino acid sequence of the rat lanosterol synthase exhibits 41.1%, 38.3% and 45.3% identity to those of the oxidosqualene cyclases of yeast, Candida albicans and Arabidopsis thaliana, respectively. Taking advantage of this homology, human liver lanosterol synthase gene was cloned by PCRs. The obtained clone was successfully expressed in yeast as active enzyme.
