C-24-methylation of 26-fluorocycloartenols by recombinant sterol C-24-methyltransferase from soybean: evidence for channel switching and its phylogenetic implications

Abstract

The tightly coupled nature of the electrophilic alkylation reaction sequence catalysed by 24-SMT (sterol C-24-methyltransferase) of land plants and algae can be distinguished by the formation of cationic intermediates that yield phyla-specific product profiles. C-24-methylation of the cycloartenol substrate by the recombinant Glycine max (soybean) 24-SMT proceeds to a single product 24(28)-methylenecycloartanol, whereas the 24-SMT from green algae converts cycloartenol into two products cyclolaudenol [∆25(27)-olefin] and 24(28)-methylenecycloartanol [∆24(28)-olefin]. Substrate analogues that differed in the steric-electronic features at either end of the molecule, 26-homocycloartenol or 3β-fluorolanostadiene, were converted by G. max SMT into a single 24(28)-methylene product. Alternatively, incubation of the allylic 26-fluoro cyclosteroid with G. max SMT afforded a bound intermediate that converted in favour of the ∆25(27)-olefin product via the cyclolaudenol cation formed initially during the C-24-methylation reaction. A portion of the 26-fluorocycloartenol substrate was also intercepted by the enzyme and the corresponding hydrolysis product identified by GC-MS as 26-fluoro-25-hydroxy-24-methylcycloartanol. Finally, the 26-fluorocycloartenols are competitive inhibitors for the methylation of cycloartenol and 26-monofluorocycloartenol generated timedependent inactivation kinetics exhibiting a kinact value of 0.12 min−1. The ability of soybean 24-SMT to generate a 25-hydroxy alkylated sterol and fluorinated ∆25(27)-olefins is consistent with our hypothesis that (i) achieving the cyclolaudenyl cation intermediate by electrophilic alkylation of cycloartenol is significant to the overall reaction rate, and (ii) the evolution of variant sterol C-24-methylation patterns is driven by competing reaction channels that have switched in algae from formation of primarily ∆25(27) products that convert into ergosterol to, in land plants, formation of ∆24(28) products that convert into sitosterol.