Isolation and characterization of an active-site peptide from a monoterpene cyclase labeled with a mechanism-based inhibitor.
(+)-Pinene synthase and (+)-bornyl pyrophosphate synthase from culinary sage (Salvia officinalis L.: Lamiaceae) catalyze the coupled isomerization and cyclization of geranyl pyrophosphate to the indicated bicyclic monoterpenes. The reaction parameters for these monoterpene cyclases have been well defined but the two enzymes have proved difficult to separate and purify in sufficient amounts for detailed structural characterization. A method was developed for the isolation of the two cyclases from highly enriched sage leaf oil gland extracts and for the efficient copurification of the two enzymes to about 95% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); the preparation yielded two overlapping species on two-dimensional polyacrylamide gel electrophoresis as expected. The cyclases were copurified and labeled with the highly selective mechanism-based irreversible inactivator 6-[1-3H]cyclopropylidene-3E-methyl-hex-2-en-1-yl pyrophosphate, subjected to cleavage with CNBr, and the resulting covalently modified peptides were isolated by SDS-PAGE for blotting to a polyvinylidene difluoride membrane and N-terminal amino acid sequence analysis. A radiochemically abundant 5-kDa peptide of the cleavage mixture was shown to be highly homologous, through 22 residues, to a segment (Leu197-Glu218) of (-)-4S-limonene synthase from spearmint (Mentha spicata L.: Lamiaceae), the only monoterpene cyclase for which the complete deduced amino acid sequence is known. These results illustrate the use of the mechanism-based inhibitor for purification and structural studies with the monoterpene cyclases, and they define a presumptive active site region that bears a highly conserved sequence among these enzymes from the mint (Lamiaceae) family.
McGeady
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P
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Croteau
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D019753:Intramolecular Lyases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology; D016415:Sequence Alignment
D007535:Isomerases; D019753:Intramolecular Lyases; C040265:pinene cyclase I