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 24 beta-methyl/ethyl Delta(25(22))-olefin products typical of primitive organisms. Unnatural Delta(24(25))-sterol substrates, missing a C4 beta-angular methyl group involved with binding orientation, convert to product ratios in favor of Delta(24(28))-products. Remodeling the active site to alter the electronics of Tiy110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Delta(24)-bond, that thereby produces metabolic switching of product ratios in favor of Delta(25(27))-olefins or impairs the second C-1-transfer activity. Incubation of [27-C-13]lanosterol or [methyl-H-2(3)]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the "algal" Delta(25(27))-olefin route, where methylation proceeds by a conserved S(N)2 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 (24 beta-methyl) biosynthesis to stigmasterol (24 alpha-ethyl) biosynthesis during the course of land plant evolution. (C) 2014 Elsevier Ltd. All rights reserved.