Structural requirements for transformation of substrates by the (S)-aenosyl-L-methionine:Δ²⁴⁽²⁵⁾-sterol methyl transferase

The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bisubstrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the microsomal (S)adenosyl-L-methionine:sterol Δ²⁴-methyl transferase (SMT) have been established for the first time. The microsomal preparation was found to catalyze the formation of a Δ²⁴⁽²⁸⁾-sterol and to be free of contaminating methyl transferase enzymes, e.g. those which form Δ^23-24 methyl sterols (cyclosadol) and Δ^25-24β-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate. From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT. In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates. Of the chemical groups which distinguishes cycloartenol, a free 3β-OH,9β,19-cyclopropyl group, trimethylated saturated nucleus, and Δ²⁴-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation. Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. (1988) Proc. Natl. Acad. Sei. U. S. A. 85, 5759-5763).