title: Active site mapping of affinity-labeled rat oxidosqualene cyclase.
abstract: Rat liver oxidosqualene cyclase (OSC), a 78-kDa membrane-bound enzyme, was purified and labeled with the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene (Abe, I., Bai, M., Xiao, X.-Y., and Prestwich, G. D. (1992) Biochem. Biophys. Res. Commun. 187, 32-38). A 6-kDa CNBr peptide was separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride membrane. The sequence of the first 30 amino acids of this peptide were determined by Edman degradation and showed unexpectedly high similarity to the fungal OSC from Candida albicans (50% identity with Arg413-Val442) and to the bacterial squalene cyclase from Alicyclobacillus (formerly Bacillus) acidocaldarius (37% identity with Lys356-Leu385). Further, radioanalysis clearly established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala or DDTAEA) were equally labeled by the irreversible inhibitor. This result provides the first information on the structural details of the active site of OSC and shows for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of sesquiterpene cyclases and prenyl transferases.
authors.affiliation: Department of Chemistry, University at Stony Brook, New York 11794-3400.
authors.forename: I
authors.initials: I
authors.lastname: Abe
authors.forename: G D
authors.initials: GD
authors.lastname: Prestwich
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019751:Intramolecular Transferases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D000345:Affinity Labels; D010446:Peptide Fragments; D007535:Isomerases; D019751:Intramolecular Transferases; C021924:lanosterol synthase