Characterization, mutagenesis and mechanistic analysis of an ancient algal sterol C24-methyltransferase: Implications for understanding sterol evolution in the green lineage.

Sterol C24-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the pattern of sterol diversity across eukaryotic kingdoms. The only gene for sterol alkylation in green algae was identified and the corresponding catalyst from Chlamydomonas reinhardtii (Cr) was characterized kinetically and for product distributions. The properties of CrSMT were similar to those predicted for an ancient SMT expected to possess broad C3-anchoring requirements for substrate binding and formation of 24ß-methyl/ethyl Δ(25(27))-olefin products typical of primitive organisms. Unnatural Δ(24(25))-sterol substrates, missing a C4ß-angular methyl group involved with binding orientation, convert to product ratios in favor of Δ(24(28))-products. Remodeling the active site to alter the electronics of Try110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Δ(24)-bond, that thereby produces metabolic switching of product ratios in favor of Δ(25(27))-olefins or impairs the second C1-transfer activity. Incubation of [27-(13)C]lanosterol or [methyl-(2)H3]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the "algal" Δ(25(27))-olefin route, where methylation proceeds by a conserved SN2 reaction and de-protonation proceeds from the pro-Z methyl group on lanosterol corresponding to C27. This previously unrecognized catalytic competence for an enzyme of sterol biosynthesis, together with phylogenomic analyses, suggest that mutational divergence of a promiscuous SMT produced substrate- and phyla-specific SMT1 (catalyzes first biomethylation) and SMT2 (catalyzes second biomethylation) isoforms in red and green algae, respectively, and in the case of SMT2 selection afforded modification in reaction channeling necessary for the switch in ergosterol (24ß-methyl) biosynthesis to stigmasterol (24α-ethyl) biosynthesis during the course of land plant evolution.

Sterol C24-methyltransferase: Physio- and stereo-chemical features of the sterol C3 group required for catalytic competence.

Sterol C24-methyltransferases (24-SMTs) catalyze the electrophilic alkylation of Δ(24)-sterols to a variety of sterol side chain constructions, and the C3- moiety is the primary determinant for substrate binding by these enzymes. To determine what specific structural features of the C3-polar group ensure sterol catalysis, a series of structurally related C3-analogs of lanosterol that differed in stereochemistry, bulk and electronic properties were examined against the fungal 24-SMT from Paracoccidioides brasiliensis (Pb) which recognize lanosterol as the natural substrate. Analysis of the magnitude of sterol C24-methylation activity (based on the kinetic constants of V(max)/K(m) and product distributions determined by GC-MS) resulting from changes at the C3-position in which the 3ß-OH was replaced by 3α-OH, 3ß-acetyl, 3-oxo, 3-OMe, 3ß-F, 3ß-NH(2) (protonated species) or 3H group revealed that lanosterol and five substrate analogs were catalyzed and yielded identical side chain products whereas neither the 3H- or 3α-OH lanosterol derivatives were productively bound. Taken together, our results demonstrate a chemical complementarity involving hydrogen bonding formation of specific active site contacts to the nucleophilic C3-group of sterol is required for proper orientation of the substrate C-methyl intermediate in the activated complex.