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 Δ25(27)-olefin products typical of primitive organisms. Unnatural Δ24(25)-sterol substrates, missing a C4β-angular methyl group involved with binding orientation, convert to product ratios in favor of Δ24(28)-products. Remodeling the active site to alter the electronics of Try110 (to Leu) results in delayed timing of the hydride migration from methyl attack of the Δ24-bond, that thereby produces metabolic switching of product ratios in favor of Δ25(27)-olefins or impairs the second C1-transfer activity. Incubation of [27-13C]lanosterol or [methyl-2H3]SAM as co-substrates established the CrSMT catalyzes a sterol methylation pathway by the “algal” Δ25(27)-olefin route, where methylation proceeds by a conserved SN2 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 (24β-methyl) biosynthesis to stigmasterol (24α-ethyl) biosynthesis during the course of land plant evolution.
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...
The objective of this study was to determine how a high-intensity circuit-training (HICT) program affects key physiological health markers in sedentary obese men. Eight obese (body fat percentage >26%) males completed a four-week HICT program, consisting of three 30-minute exercise sessions per week, for a total of 6 hours of exercise. Participants' heart rate (HR), blood pressure (BP), rating of perceived exertion, total work (TW), and time to completion were measured each exercise session, body composition was measured before and after HICT, and fasting blood samples were measured before throughout, and after HICT program. Blood sample measurements included total cholesterol, triacylglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, glucose, and insulin. Data were analyzed by paired t-tests and one-way ANOVA with repeated measures. Statistical significance was set to P < 0.05. Data analyses revealed significant (P < 0.05) improvements in resting HR (16% decrease), systolic BP (5.5% decrease), TW (50.7%), fat tissue percentage (3.6%), lean muscle tissue percentage (2%), cholesterol (13%), triacylglycerol (37%), and insulin (18%) levels from before to after HICT program. Overall, sedentary obese males experienced a significant improvement in biochemical, physical, and body composition characteristics from a HICT program that was only 6 hours of the total exercise.
Abbreviations: AZA, 25-azalanosterol; BSF, bloodstream form; DOX, doxycycline; ED, effective dose; FGM, full-growth medium; ITC, itraconazole; LDM, lipid-depleted medium; PCF, procyclic form; RRTc, relative retention time with cholesterol used as standard; SAM, S-adenosyl-lmethionine; SDM, sterol C14-demethylase; SMT, sterol C24-methyltransferase; Tb SDM, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C14-demethylase; Tb SMT, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C24-methyltransferase; TetR, tetracycline repressor .
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