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 24β-methyl/ethyl Δ(25(27))-olefin products typical of primitive organisms.

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 3β-OH was replaced by 3α-OH, 3β-acetyl, 3-oxo, 3-OMe, 3β-F, 3β-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.