Sterol C-methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition

The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca,w ickerhamii, a non-photosynthetic, yeast-like alga, was found to C-methylate appropriate Delta(24(25))-sterol acceptor molecules to Delta(25(27))-24 be ta-methyl products stereoselectively. Incubation with pairs of substrates-[ H-2(3)-methyl]AdoMet and cycloartenol, and AdoMet and [27(-13)C]lanosterol- followed by H-1 and C-13 NMR analysis of the isotopically labeled products demonstrated the si-face (beta-face attack) mechanism of C-methylation and the regiospecificity of Delta(25(27))-double bond formation from the pro-Z methyl group (C27) on lanosterol. The enzyme has a substrate preference for a sterol with a 3 beta-hydroxyl group, a planar nucleus and a side chain o riented into a 'right-handed' structure (20R-chirality)-characteristic of t he native substrate, cycloartenol. The apparent native molecular weight of the SMT was determined to be approximately 154,000, as measured by Superose 6 FPLC. A series of sterol analogues which contain heteroatoms substituted for C24 and C25 or related structural modifications, including steroidal a lkaloids, havs been used to probe further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modif ications of a positively charged moiety in the form of an ammonium group su bstituted for carbon at C25, C24, C23 or C22 are particularly potent non-co mpetitive inhibitors (K-i for the most potent inhibitor tested, 25-azacyclo artanol, was ca. 2nM, four orders of magnitude less than the K-m for cycloa rtenol of 28 mu M), supporting the intermediacy of the 24-methyl C24(25)-ca rbenium ion intermediate. Ergosterol, but neither cholesterol nor sitostero l, was found to inhibit SMT activity (K-i = 80 mu M). The combination of re sults suggests that the interrelationships of substrate functional groups w ithin the active center of a Delta(24(25)) to Delta(25(27)) 24 beta-methyl- SMT could be approximated thereby allowing the rational design of C-methyla tion inhibitors to be formulated and tested. (C) 2000 Elsevier Science Ltd. All rights reserved.