METHODOLOGY FOR THE PREPARATION OF PURE RECOMBINANT SACCHAROMYCES-CEREVISIAE LANOSTEROL SYNTHASE USING A BACULOVIRUS EXPRESSION SYSTEM - EVIDENCE THAT OXIRANE CLEAVAGE AND A-RING FORMATION ARE CONCERTED IN THEBIOSYNTHESIS OF LANOSTEROL FROM 2,3-OXIDOSQUALENE

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanoster ol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae wh ich catalyzes the complex cyclization/rearrangement step in sterol bio synthesis, was overexpressed in baculovirus-infected cells and purifie d to homogeneity in three steps. Using pure enzyme the kinetics of cyc lization were determined using Michaelis-Menten analysis for 2,3-oxido squalene (1) and two analogs in which the C-6 methyl was replaced by H (3) or Cl (4). The measured V-max/K-M ratios for 1, 3, and 4 were fou nd to be 138, 9.4, and 21.9, respectively, a clear indication that oxi rane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate o f oxirane cleavage. No catalytic metal ions could be detected in purif ied lanosterol synthase by atomic absorption analysis. Site-directed m utagenesis studies of each of the six strongly conserved aspartic acid residues (D --> N mutation) and each of the nine conserved glutamic a cid residues (E --> Q) revealed that only one, D456, is essential for catalytic function of the enzyme. The essential D456 residue is a like ly candidate for electrophilic (specifically protic) activation of the oxirane function.