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

Abstract The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii , a non-photosynthetic, yeast-like alga, was found to C -methylate appropriate Δ 24(25) -sterol acceptor molecules to Δ 25(27) -24β-methyl products stereoselectively. Incubation with pairs of substrates—[ 2 H 3 - methyl ]AdoMet and cycloartenol, and AdoMet and [27- 13 C]lanosterol—followed by 1 H and 13 C NMR analysis of the isotopically labeled products demonstrated the si -face (β-face attack) mechanism of C -methylation and the regiospecificity of Δ 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 3β-hydroxyl group, a planar nucleus and a side chain oriented into a ‘right-handed’ structure (20 R -chirality)—characteristic of the 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 alkaloids, havs been used to probe further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modifications of a positively charged moiety in the form of an ammonium group substituted for carbon at C25, C24, C23 or C22 are particularly potent non-competitive inhibitors ( K i for the most potent inhibitor tested, 25-azacycloartanol, was ca. 2 nM, four orders of magnitude less than the K m for cycloartenol of 28 μM), supporting the intermediacy of the 24-methyl C24(25)-carbenium ion intermediate. Ergosterol, but neither cholesterol nor sitosterol, was found to inhibit SMT activity ( K i =80 μM). The combination of results suggests that the interrelationships of substrate functional groups within the active center of a Δ 24(25) to Δ 25(27) 24β-methyl-SMT could be approximated thereby allowing the rational design of C -methylation inhibitors to be formulated and tested.