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.
Highlights
Peating motif rich in aromatic amino acids? suggesting that vertebrate and fungal OSC evolved from the prokaryotic bacterial SCs
Stony Brook, New York 11794-3400 (201, covalently modified enzymatically active OSCs from rat, Rat liver oxidosqualene cyclase (OSCa),78-kDa membrane-bound enzyme, was purified and labeled with pig, dog, and theaech species human liver, identifying single protein with molecular masseras nging from 70 bands in to80 kDa mechanism-based irreversible inhibitor, [sH]29-methyl- [8].The mechanism of inhibition required substrate turnover idene-2,3-oxidosqualene
The sequence of the first 30 amino acids of had been modified by tritium-labeled 29-M(OlbS,T = 3H). The this peptide were determined by Edman degradation active site of rat liver OSC affinity labeled by this suicide suband showed unexpectedly high similarityto the fungal strate occurred in one of the conserved regions found in the OSCfrom Canclidcr albicans
Summary
CDNAsfor the C. albicans OSC and two bacterial squalene cyclases (SCs) revealedhighlyconservedregionswith a re-. (201, covalently modified enzymatically active OSCs from rat, Rat liver oxidosqualene cyclase (OSCa),78-kDa membrane-bound enzyme, was purified and labeled with pig, dog, and theaech species human liver, identifying single protein with molecular masseras nging from 70 bands in to kDa mechanism-based irreversible inhibitor, [sH]29-methyl- [8].The mechanism of inhibition required substrate turnover idene-2,3-oxidosqualene The sequence of the first 30 amino acids of had been modified by tritium-labeled 29-M(OlbS,T = 3H). The this peptide were determined by Edman degradation active site of rat liver OSC affinity labeled by this suicide suband showed unexpectedly high similarityto the fungal strate occurred in one of the conserved regions found in the OSCfrom Canclidcr albicans Val44a)and to the bacterial squalene cyclase froAmlicy- that was labeled is shared by two other enzyme families clobacillus (formerly Bacillusa) cidocaldarius (37% important in the terpene biogenesis,the sesquiterpene cyclases identitywith Lyss~-Leus~s)F.urtherr,adioanalysis and the prenyl transferases. clearly established that the two adjacentAsp residues in thehighlyconservedregion(Asp-Asp-Thr-Ala-Glu-
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