Studies on the purification and characterization of the (s)-adensyl-l-methionine sterol methyl transferase from Arabidopsis thaliana

The sterol methyl transferase gene from Arabidopsis thalicma that encodes for the 24 consecutive C-methylation of the A -bond of the sterol side chain was introduced into plasmid pET15b and the resuhing native protein was overexpressed in BL2i(DE3) host cells under control of a T? promoter. The enzyme was partially purified by anion exchange and gel filtration chromatography. GPC chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a native molecular weight of 158.000 Dahons and was tetrameric. Studies on product formation using sterols native to A. thaliana, cycloartenol (K^^^^^^ = 46 ^M and y^^^^ppj = ^ pmo/min/mg) and 24(28)-methylene lophenol 0^m{app) " ^^ I^^ ^"^^maxíapp) ^ ^-^^ pmol/min/mg), as substrates incubated with a soluble SMT preparation confirmed that the first and second C-methyl transfer reactions proceeded stereoselctively to a single product 24(28)-methyIene cycloartanol and 24(28)Z-thyIidene lophenol, respectively. From a study involving incubation of a series of related substrates that were structurally similar to cycloartenol and 24(28)methylene lophenol with a soluble enzyme preparation of the SMT, the sterol features recognized by the SMT for binding were established: a free 3P-hydroxyl group, a side chain with a A" -bond and a planar nucleus. An unexpected fmding was that zymosterol (K^(Û„„; ^28 |iM and yjnnxfappj ^11^ pmol/min/mg), the preferred sterol substrate for the yeast SMT was more effective in binding to the plant SMT from A. thaliana than cycloartenol. From a kinetic study involving a series of high energy intermediate and product analogs, we observed that 24p-methyl sterols inhibited Cmethylation consistent whh the proposed mechanism of a iS/-face attack ("Steric-EIectric Plug model" and that the first and second C-methyl transfer reactions utilize different substrates, but nonetheless undergo transformation from the same binding site. Sitosterol, but not cholesterol was bound productively to the SMT and inhibited enzyme activity with competitive-type kinetics relative to ehher cycloartenol or 24(28)-methyIene lophenol as substrates. Using a ftision construct containing a His Tag and a native construct, a set of shedirected mutants have been generated at Trp89 and Tyr85, aromatic amino acid residues assumed to be contained whhin the sterol binding site of this enzyme. Whereas no change in ehher the product distribution or sterol specificity of these mutants was observed, a change in catalytic efficiences was established in several cases. From the combination of resuhs, the interrelationships of substrate fiinctional groups whhin the active center could be approximated showing that a single SMT can give rise to mono or doubly alkylated side chains by the same reaction mechanism.