Biosynthesis of Phytosterols

Abstract

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C 1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s ؊1 , respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-L-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. H 3 ]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the ⌬ 24 bond.Sterol methyltransferases (SMTs) 1 are ubiquitously represented in plants and fungi (1). Together, these enzymes generate 24-alkyl sterol diversity, which includes formation of singly and doubly C-24-alkylated sterol side chains and olefin variants possessing ⌬ 24(28) , ⌬ 23 (24) , and ⌬ 25(27) side chains (2). In most organisms, SMTs catalyze the first committed step in the biosynthesis of phytosterols (3, 4) (Fig. 1). The crucial role of these enzymes to generate an essential physiological group in sterol structure has stimulated considerable interest in the stereochemistry and mechanism of the C-methylation reaction (5, 6). Several SMTs have been characterized at the molecular level, and their amino acid compositions reveal a highly conserved signature motif that represents the sterol-binding site (7,8). A number of these enzymes have been characterized, and they share similar native molecular masses in the range of 160 -175 kDa and similar properties (1). The methyl transfer reaction catalyzed by SMT is proposed to proceed through a nucleophilic attack by the electrons of the ⌬ 24 double bond on the S-methyl group of AdoMet (9 -11). The reaction can lead to the formation of a high energy intermediate (HEI) possessing a methyl at C-24 and a carbonium ion at C-25. After a hydride transfer from C-24 to C-25, an elimination of a proton at C-28 occurs, giving a 24(28)-methylene sterol. The steric course of the reaction has been hypothesized to proceed by an "X-group" (covalent), carbocation, or concerted mechanism (Scheme 1) (8).As recognized in the steric-electric plug model and X-group mechanism, the conformation of the bound sterol side chain can influence the configuration of the enzyme-generated product at C-24 and C-25 (Scheme 1A) (8).Recent work on the stereochemistry of phytosterol (24-alkyl sterols) side chain carbon atoms harboring chemically or biosynthetically introduced 13 C label showed that the natural configuration for C-26 and C-27 of ergosterol and sitosterol (C-25 S; 2) is opposite to that...

Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion

“…Thus, plasma membrane proteins tended to accumulate in other fractions such as IM and SP. Even though the loss of smt1 function shows a pleiotropic whole plant phenotype (20,21,64,73 ), a ratio based analysis, using the Unicorn algorithm, allows to draw conclusions specifically on proteinsterol interactions. General protein abundance levels, membrane trafficking and internalization processes of proteins might be affected, but analyzing DRM and DSF fractions in parallel, focused the analysis on to the relative distribution of plasma membrane incorporated proteins.…”

Mechanistic Analysis of a Multiple Product Sterol Methyltransferase Implicated in Ergosterol Biosynthesis in Trypanosoma brucei

“…Substrates and Reagents-The preparation, proof of structure, and purification of 26,27-dehydrozymosterol, 25-azalanosterol, 24(R,S),25-epiminolanosterol, and ergosterol have been described (9,(13)(14)(15). Sterol substrates for activity assay were from our steroid collection (13)(14)(15) Trypanosoma brucei SMT Cloning, Expression, and Purification-Sequence data for T. brucei SMT were obtained from the web site of The Institute of Genomic Research (www.tigr.org) using Blast search against SMT from S. cerevisiae.…”

“…However, using cloned enzymes to study product profiles as well as deuterium isotope effects on these enzymatic reactions assayed with native enzymatic preparations has indicated that a single SMT enzyme can produce mixtures of mono-methyl(ene) or di-methyl(ene) sterol products (12)(13)(14)(15). SMTs are presently categorized mechanistically based on substrate specificity and product distribution as: 1) SMT1, which catalyzes the first C 1 -transfer reaction of cycloartenol to 24 (28)-methylene cycloartanol (EC.2.1.1.143, e.g.…”

“…Saccharomyces cerevisiae); or 3) SMT2, which catalyzes the second C 1 -transfer reaction of 24 (28)-methylene lophenol to a mixture of 24 (28)Z-ethylidene lophenol, 24 (28)E-ethylidene lophenol, and 4␣-methyl 24␤-ethyl cholesta-7, 25 (27)-dienol (EC.2.1.1.143, e.g. Arabidopsis thaliana) (13)(14)(15)(16).…”

Muriceanol, a 24(28)‐Epoxide Sterol Link in the Carbon Flux Toward Side‐Chain Dealkylation of Sterols

“…The C-24 alkyl group containing phytosterols come from S-adenosyl-l-methionine [12] and C-methylation is an energy-expensive process costing the cell about 14 ATP per methyl group. [13] Therefore, it would be a big paradox that Muricea spp biosynthesizes 24-methylenecholesterol (5) from desmosterol (4; Scheme 1) and dealkylates 24-methylenecholesterol (5) via epoxide intermediate 1 as an extra pathway to obtain 6.…”