Overexpression, Purification, and Stereochemical Studies of the Recombinant (S)-Adenosyl-l-methionine: Δ24(25)- to Δ24(28)-Sterol Methyl Transferase Enzyme fromSaccharomyces cerevisiae

Nes, W. David; Mccourt, B. S.; Zhou, Wen; Ma, Jie; Marshall, Julie A.; Peek, L.-A.; Brennan, Michael

doi:10.1006/abbi.1998.0665

Cited by 50 publications

(65 citation statements)

References 43 publications

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“…We used this source of plant SMT to provide preliminary characterization of the first and second C 1 transfer activities; the enzymegenerated products were characterized by a combination of MS and 1 H NMR (31). Purification of the soybean SMT was accomplished with a modified protocol established previously for the purification of the yeast SMT (20), as summarized in Table I.…”

Section: Resultsmentioning

confidence: 99%

“…The liberated SMT gene was then ligated into pET23a using the NdeI and BamHI overhangs generated during digestion. The resulting ligation mixture was transformed into BL21(DE3)-competent E. coli cells by heat shock (20). Sequencing confirmed that no errors had been introduced by the polymerase reactions (dideoxy terminator sequencing ABI 373 sequencer).…”

Section: Methodsmentioning

confidence: 97%

“…The incubation mixture was terminated with 500 l of a solution of 10% methanolic KOH. The methylated sterol product was extracted three times with 2.5 ml each in hexane (Fisher) and mixed on a vortex mixer for 30 s. The resulting organic layer was then transferred to a 7-ml scintillation vial, and the sample was taken for liquid scintillation counting to determine conversion rate (20). For kinetic analysis, standard assays were performed at 10 substrate concentrations from 5 to 200 M sterol with the AdoMet concentration held at saturation of 50 M. The K m and V max for AdoMet were determined from 5 to 200 M with sterol concentration held at 50 M. From the variation of reaction velocity with substrate concentration according to Michaelis-Menten kinetics, an apparent saturation of either substrate approached 50 M (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…Previously published kinetics for SMT relied on a microsomebound or soluble enzyme, which we now know can contain a low abundance of enzyme with significant substrate contaminants (9, 10, 20, 33). However, the use of recombinant SMT overexpressed in E. coli (20) has enabled the estimation of actual enzyme concentrations and, thereby, the comparison of exacting kinetic parameters for the first time.…”

Section: Conversion Of 28-2 H Substrates To 24-methylatedmentioning

confidence: 99%

“…SMT2 is considered to monomethylate the product of the first C 1 transfer reaction, forming ⌬ 24 (28) Z-ethylidene sterols exclusively. Nes et al (20) purified the yeast SMT to homogeneity in 1998 and generated a mutant by site-directed mutagenesis, making it clear that both methyl transfer reactions involving ⌬ 24 (25) -and ⌬ 24(28) -sterols can be catalyzed by a single enzyme and that the second C 1 transfer activity can result from the same active site as the first C 1 transfer activity to give multiple products. However, as of yet, the overall catalytic action of a plant SMT has not been established.…”

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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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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Conversion Of 28-2 H Substrates To 24-methylatedmentioning

confidence: 99%

mentioning

confidence: 99%

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Biosynthesis of Phytosterols

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Song

Dennis

et al. 2003

Journal of Biological Chemistry

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Methylierung nicht‐aktivierter Alkene mit modifizierten Methyltransferasen zur Diversifizierung von Terpenoiden

et al. 2023

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Terpenoide sind aus Isopren‐Bausteinen aufgebaut und haben zahlreiche biologische Funktionen. Eine selektive Modifizierung ihres Kohlenstoffgerüsts hat das Potenzial die biologischen Aktivitäten zu verändern oder zu optimieren. Die Synthese von Terpenoiden mit einem nicht‐natürlichen Kohlenstoffgerüst ist jedoch aufgrund der Komplexität dieser Moleküle oft ein schwieriges Unterfangen. In dieser Arbeit berichten wir über die Identifizierung und das Engineering von (S)‐Adenosyl‐l‐Methionin‐abhängigen Sterol‐Methyltransferasen für die selektive C‐Methylierung von linearen Terpenoiden. Das entwickelte Enzym katalysiert die selektive Methylierung von nicht‐aktivierten Alkenen in Mono‐, Sesqui‐ und Diterpenoiden zur Herstellung von C11‐, C16‐ und C21‐Derivaten. Die präparative Umsetzung und Isolation der Produkte zeigt, dass dieser Biokatalysator die Bildung von C−C‐Bindungen mit hoher Chemo‐ und Regioselektivität durchführt. Die Methylierung des Alkens erfolgt höchstwahrscheinlich über ein Carbokation‐Zwischenprodukt und eine regioselektive Deprotonierung. Diese Methode eröffnet neue Wege zur Modifizierung des Kohlenstoffgerüsts von Alkenen im Allgemeinen und Terpenoiden im Besonderen.

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Methylation of Unactivated Alkenes with Engineered Methyltransferases To Generate Non‐natural Terpenoids

et al. 2023

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Terpenoids are built from isoprene building blocks and have numerous biological functions. Selective late-stage modification of their carbon scaffold has the potential to optimize or transform their biological activities. However, the synthesis of terpenoids with a non-natural carbon scaffold is often a challenging endeavor because of the complexity of these molecules. Herein we report the identification and engineering of (S)-adenosyl-L-methionine-dependent sterol methyltransferases for selective C-methylation of linear terpenoids. The engineered enzyme catalyzes selective methylation of unactivated alkenes in mono-, sesquiand diterpenoids to produce C 11 , C 16 and C 21 derivatives. Preparative conversion and product isolation reveals that this biocatalyst performs CÀ C bond formation with high chemo-and regioselectivity. The alkene methylation most likely proceeds via a carbocation intermediate and regioselective deprotonation. This method opens new avenues for modifying the carbon scaffold of alkenes in general and terpenoids in particular.

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Overexpression, Purification, and Stereochemical Studies of the Recombinant (S)-Adenosyl-l-methionine: Δ24(25)- to Δ24(28)-Sterol Methyl Transferase Enzyme fromSaccharomyces cerevisiae

Cited by 50 publications

References 43 publications

Biosynthesis of Phytosterols

Biosynthesis of Phytosterols

Methylierung nicht‐aktivierter Alkene mit modifizierten Methyltransferasen zur Diversifizierung von Terpenoiden

Methylation of Unactivated Alkenes with Engineered Methyltransferases To Generate Non‐natural Terpenoids

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