Stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation in ergosterol biosynthesis

Zhou, Wen; Guo, Dean; Nes, W. David

doi:10.1016/0040-4039(96)00017-2

Cited by 23 publications

(17 citation statements)

References 17 publications

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“…In his seminal report, Arigoni defined the stereochemistry at C-25 in fungal ergosterol to be 25S [32]. We confirmed that ergosterol and sitosterol are the methylene products of SMT1 and that they possessed the 25S stereochemistry [33][34][35] which is distinct from that of animal cholesterol which assumes the 25R stereochemistry following reduction of the 24, 25-double bond [36]. The stereochemistry at C-25 is retained during the second alkylation step [37].…”

Section: Mechanisms Of Catalysismentioning

confidence: 52%

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

et al. 2004

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Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.

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Section: Mechanisms Of Catalysismentioning

confidence: 52%

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

et al. 2004

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“…The demonstration of the expected number of deuterium atoms incorporated in the enzyme-generated product and chemical reasonableness proves that the deuterium-labeled substrate actually was involved with the catalysis. The position of the deuterium label at C-28 or C-25 in the respective enzyme-generated product was confirmed by 1 H NMR (500 MHz) analysis against reference specimens (13,25). The proposed stereochemical model for the C-methylation of cycloartenol to 24(28)-methylenecycloartanol predicts that the methyl donation to ⌬ 24 occurs from the si (␤)-face of the substrate double bond (Scheme 1A) (8,12).…”

Section: Resultsmentioning

confidence: 90%

“…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 considered in the X-group and carbonium ion mechanisms (C-25 R; 2a) (11)(12)(13) and therefore suggested a general catalytic mechanism for sterol C-methylation. Early kinetic studies carried out with microsomal preparations or detergent-solubilized enzyme preparations ruled out a Ping Pong (covalent) mechanism and supported either a concerted or carbocation mechanism for catalysis (9,10).…”

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confidence: 99%

“…Support for the carbocation mechanism was considered to result from the intermediacy of HEIs in sterol C-methylation catalysis, whereby mimics of the transition state species were effective inhibitors of plant and fungal SMT activity (1). However, the carbocation mechanism was recently eliminated as a possible route in ergosterol synthesis (operating the first C 1 transfer reaction) by work on the stereochemistry of the fungal enzyme-mediated CH 3 to CH 2 reaction and the stereochemistry at C-25 resulting from the 1,2-hydride shift reaction (11)(12)(13).…”

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confidence: 99%

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Biosynthesis of Phytosterols

Nes

Song

Dennis

et al. 2003

Journal of Biological Chemistry

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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...

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“…[27- 13 C]Lanosterol (99% 13 C) was prepared as described previously [38]. [ methyl - 3 H 3 ]AdoMet ( S -adenosylmethionine; 10–15 Ci/mmol), diluted to 10 µCi/µmol for the activity assays, was purchased from PerkinElmer.…”

Section: Methodsmentioning

confidence: 99%

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

Nes

Singha

Liu

et al. 2012

Biochemical Journal

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Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14-demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate–mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control <[2-13C]leucine<[2-13C]acetate<[1-13C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.

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Stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation in ergosterol biosynthesis

Cited by 23 publications

References 17 publications

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

Biosynthesis of Phytosterols

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

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