Active site mapping of affinity-labeled rat oxidosqualene cyclase

• Published in: The Journal of biological chemistry Vol. 269; no. 2; pp. 802 - 804
• Main Authors: ABE, I, PRESTWICH, G. D
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 14.01.1994
• Subjects: Affinity Labels; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Intramolecular Transferases; Isomerases - antagonists & inhibitors; Isomerases - chemistry; Liver - enzymology; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Mapping; Rats; Sequence Alignment; Sequence Homology, Amino Acid; Transferases; Radiolabelling; Rat; Enzyme; Affinity labelling; Active site; Rodentia; Enzyme inhibitor; Vertebrata; Isomerases; Mammalia; Intramolecular transferases; Lanosterol synthase; Comparative study; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)42179-x

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.