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W. David Nes - One of the best experts on this subject based on the ideXlab platform.
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Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans.
Archives of biochemistry and biophysics, 2010Co-Authors: Kulothungan Ganapathy, Ragu Kanagasabai, Thi Thuy Minh Nguyen, W. David NesAbstract:Abstract Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for Zymosterol with associated kinetic constants of Km 50 μM and kcat of 0.01 s−1. The product of the enzyme incubated with Zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, Ki = 54 nM and 24 (R,S),25-epiminolanosterol, Ki = 11 nM) or as mechanism-based inactivators (26,27-dehydroZymosterol, Ki 9 μM) and kinact = 0.03 min−1) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording Ki values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC50 values that ranged from 3 to 20 μM.
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Yeast sterol C24-methyltransferase: role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis.
Archives of biochemistry and biophysics, 2008Co-Authors: W. David Nes, Pruthvi Jayasimha, Zhihong SongAbstract:Abstract The role of Try-81 in the reaction catalyzed by Saccharomyces cerevisiae sterol 24-C-methyltransferase (Erg6p) was investigated kinetically and for product differences against a panel of position-81 mutants in which Tyr was substituted with Trp, Phe, Ile, Leu, Val and Ala. The residue chosen for mutation is one that was reported previously to accept fecosterol and yield a set 24-ethyl (idene) sterol products typical of plants, showing the amino acid residue is located close to the transient C25 carbocation intermediate in the active site. One group of mutants (aromatic) tested with the natural substrate Zymosterol accelerated the C-methylation reaction ( k cat / K m ) whereas the other group of mutants (aliphatics) decreased catalytic competence as the amino acid side chain was downsized. Mutating to aromatic and assaying with the substrate analog designed as a suicide substrate 26,27-dehydroZymosterol favored C26-monol formation, whereas mutating to the aliphatic of smaller size favored C26-diol formation (a measure of enzyme alkylation). In no case was Zymosterol converted to an intermediate that formed a C25-diol. Thermodynamic analysis (determination of E a , Δ G ‡ , Δ H ‡ and T Δ S ‡ ) for the C-methylation reaction performed by these enzymes assayed with the substrate and its analog or Zymosterol paired with the “charged’ high energy intermediate (HEI) analogs 24( R,S )25,epiminolanosterol and 25-azalanosterol or “neutral” membrane insert ergosterol showed that mutation to aromatics can reduce inhibitor potency (measured as K m / K i ), yet catalysis can improve in Trp81 by the introduction of a gain in free energy associated with stabilization of the transition state of a rate-controlling step directed toward turnover. Alternatively, mutation to the smaller aliphatic amino acid side chains led to a destabilization in the active site structure which was accompanied by increases in the partition ratios associated with abortive complex formation. The results are explained by consideration of the functional differences attributed to Tyr81 substitution to aromatics and aliphatics of different size involved with cation-π or hydrogen bonding interactions and in the activation barriers required of differing side chain conformations to orient the reactants in the direction of turnover versus enzyme inactivation.
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Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei.
The Journal of biological chemistry, 2006Co-Authors: Wenxu Zhou, Galina I. Lepesheva, Michael R. Waterman, W. David NesAbstract:Abstract Sterol methyltransferase (SMT) plays a key role in sterol biosynthesis in different pathogenic organisms by setting the pattern of the side chain structure of the final product. This catalyst, absent in humans, provides critical pathway-specific enzymatic steps in the production of ergosterol in fungi or phytosterols in plants. The new SMT gene was isolated from Trypanosoma brucei genomic DNA and cloned into an Escherichia coli expression system. The recombinant SMT was purified to homogeneity to give a band at 40.0 kDa upon SDS-PAGE and showed a tetrameric subunit organization by gel chromatography. It has a pH optimum of 7.5, an apparent kcat value of 0.01 s–1, and a Km of 47 ± 4 μm for Zymosterol. The products of the reaction were a mixture of C24-monoalkylated sterols, ergosta-8,24 (25)-dienol, ergosta-8,25 (27)-dienol, and ergosta-8,24 (28)-dienol (fecosterol), and an unusual double C24-alkylated sterol, 24,24-dimethyl ergosta-8,25 (27)-dienol, typically found in plants. Inhibitory profile studies with 25-azalanosterol (Ki value of 39 nm) or 24(R,S), 25-epiminolanosterol (Ki value of 49 nm), ergosterol (Ki value of 27 μm) and 26,27-dehydroZymosterol (Ki and kinact values of 29 μm and 0.26 min–1, respectively) and data showing Zymosterol as the preferred acceptor strongly suggest that the protozoan SMT has an active site topography combining properties of the SMT1 from plants and yeast (37–47% identity). The enzymatic activation of this and other SMTs reveals that the catalytic requirements for the C-methyl reaction are remarkably versatile, whereas the inhibition studies provide a powerful approach to rational design of new anti-sleeping sickness chemotherapeutic drugs.
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Purification, characterization and catalytic properties of human sterol 8-isomerase
Biochemical Journal, 2002Co-Authors: W. David Nes, Wenxu Zhou, Allen L. Dennis, Zhonghua Jia, Richard A. Keith, Timothy M. Piser, Stephen T FurlongAbstract:CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for Zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of Zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of Zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either Zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)
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Active site mapping and substrate channeling in the sterol methyltransferase pathway.
The Journal of biological chemistry, 2002Co-Authors: W. David Nes, Zhonghua Jia, Julie A. Marshall, Tahhan T. Jaradat, Zhihong Song, Pruthvi JayasimhaAbstract:Abstract Sterol methyltransferase (SMT) from Saccharomyces cerevisiae was purified fromEscherichia coli BL21(DE3) and labeled with the mechanism-based irreversible inhibitor [3-3H]26,27-dehydroZymosterol (26,27-DHZ). A 5-kDa tryptic digest peptide fragment containing six acidic residues at positions Glu-64, Asp-65, Glu-68, Asp-79, Glu-82, and Glu-98 was determined to contain the substrate analog covalently attached to Glu-68 by Edman sequencing and radioanalysis using C18reverse phase high performance liquid chromatography. Site-directed mutagenesis of the six acidic residues to leucine followed by activity assay of the purified mutants confirmed Glu-68 as the only residue to participate in affinity labeling. Equilibration studies indicated that Zymosterol and 26,27-DHZ were bound to native and the E68L mutant with similar affinity, whereasS-adenosylmethionine was bound only to the native SMT,K d of about 2 μm. Analysis of the incubation products of the wild-type and six leucine mutants by GC-MS demonstrated that Zymosterol was converted to fecosterol, 26,27-DHZ was converted to 26-homo-cholesta-8(9),23(24)E,26(26′)-trienol as well as 26-homocholesta-8(9),26(26′)-3β,24β-dienol, and in the case of D79L and E82L mutants, Zymosterol was also converted to a new product, 24-methylzymosta-8,25(27)-dienol. The structures of the methylenecyclopropane ring-opened olefins were determined unambiguously by a combination of 1H and 13C NMR techniques. A K m of 15 μm,K cat of 8 × 10−4s−1, and partition ratio of 0.03 was established for 26,27-DHZ, suggesting that the methylenecyclopropane can serve as a lead structure for a new class of antifungal agents. Taken together, partitioning that leads to loss of enzyme function is the result of 26,27-DHZ catalysis forming a highly reactive cationic intermediate that interacts with the enzyme in a region normally not occupied by the Zymosterol high energy intermediate as a consequence of less than perfect control. Alternatively, the gain in enzyme function resulting from the production of a Δ25(27)-olefin originates with the leucine substitution directing substrate channeling along different reaction channels in a similar region at the active site.
Martin Bard - One of the best experts on this subject based on the ideXlab platform.
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transformation of saccharomyces cerevisiae with a cdna encoding a sterol c methyltransferase from arabidopsis thaliana results in the synthesis of 24 ethyl sterols
FEBS Letters, 1996Co-Authors: Tania Husselstein, Martin Bard, Daniel Gachotte, Thierry Desprez, Pierre BenvenisteAbstract:Using an EST-cDNA probe, a full-length cDNA (411) sequence of 1411 bp was isolated from A. thaliana. This sequence contained features typical of methyltransferases in general and in particular showed 38% identity with ERG6, a S. cerevisiae gene which encodes the Zymosterol-C-24-methyltransferase. A yeast vector containing this ORF (4118-pYeDP60) was used to transform a wild type S. cerevisiae which accumulates predominantly ergosterol, a 24-methyl sterol as well as a mutant erg6 null mutant accumulating principally Zymosterol, a sterol non-alkylated at C-24. In both cases, several 24-ethyl- and 24-ethylidene sterols were synthetized indicating that the 4118 cDNA encodes a plant sterol C-methyltransferase able to perform two sequential methylations of the sterol side chain.
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A simple method for the isolation of Zymosterol from a sterol mutant of Saccharomyces cerevisiae
The Journal of steroid biochemistry and molecular biology, 1992Co-Authors: Robert W. Heidepriem, Edward J Parish, Peter Livant, Robert J. Barbuch, Maurice G. Broaddus, Martin BardAbstract:Abstract A simple method is described for the direct isolation of Zymosterol (5α-cholesta-8,24-dien-3β-ol) of high purity from a sterol mutant of Saccharomyces cerevisiae. This yeast strain, which is a double mutant of the ERG6 (sterol transmethylase) and ERG2 (C-8 sterol isomerase) genes, accumulates Zymosterol as its major sterol component.
Wen Zhou - One of the best experts on this subject based on the ideXlab platform.
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Mechanism-based active site modification of sterol methyl transferase by tritium-labeled 26-homocholesta-8,14,24-trien-26-yn-3β-ol
Tetrahedron Letters, 1998Co-Authors: W. David Nes, Julie A. Marshall, Wen Zhou, Allen L. DennisAbstract:Abstract The title compound (26-HC, 5 ) was synthesized and tested as a mechanism-based inhibitor of the sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae . Enzyme assays were performed with SMT enzyme using Zymosterol as substrate and AdoMet as coenzyme. The inhibition of SMT enzyme by 26-HC showed an apparent k i of 72.5 μM and k inact of 2.4 min −1 . Covalent modification of the active site of a SMT enzyme was demonstrated for the first time using [3- 3 H]26-HC.
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Substrate-Based Inhibitors of the (S)-Adenosyl-l-Methionine:Δ24(25)- to Δ24(28)-Sterol Methyl Transferase fromSaccharomyces cerevisiae ☆ ☆☆
Archives of biochemistry and biophysics, 1997Co-Authors: W. David Nes, De-an Guo, Wen ZhouAbstract:Abstract A series of 31 side-chain-modified analogs of cholesterol, Zymosterol, lanosterol, and cycloartenol and the steroidal alkaloids solasodine and solanidine were studied as inhibitors of ( S )-adenosyl- l -methionine:Δ 24(25) sterol methyl transferase (SMT) enzyme activity from Saccharomyces cerevisiae. Two classes of sterol methylation inhibitors were tested: substrate analogs, including mechanism-based inhibitors, and transition state analogs. Several novel sterol methylation inhibitors that contained an aza, aziridine, or ammonium group in the sterol side chain were prepared and tested for the first time. The degree and kinetic pattern of methylation inhibition were found to be influenced by the position and nature of the variant functional group introduced into the side chain. The most potent inhibitors of SMT enzyme activity were transition state analog inhibitors ( K i values of 5 to 10 n m ) that mimicked the structure and conformation of the natural substrate presumed to form in the ternary complex generated in the transition state. Steroidal alkaloids were potent competitive inhibitors with K i values ranging from 2 to 30 μ m , which is about the K m app of Zymosterol, ca. 27 μ m . An isosteric analog of the natural substrate, Zymosterol, in which the 26/27-gem-dimethyl groups were joined to form a cyclopropylidene function is shown to be a potent irreversible mechanism-based inactivator of SMT enzyme activity that exhibits competitive-type inhibition, K i 48 μ m with a K inact of 1.52 min −1 . Mechanistic implications of these results provide new insights into the topology of the ternary complex involving sterol–AdoMet–enzyme.
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Stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation in ergosterol biosynthesis
Tetrahedron Letters, 1996Co-Authors: Wen Zhou, De-an Guo, W. David NesAbstract:[methyl-2H3]Methionine and Zymosterol, [27-13C]lanosterol, [24-2H]lanosterol and lanosterol were separately incubated with the sterol auxotroph Saccharomyces cerevisiae strain GL7. Spectral evidence (1H and 13C-NMR) obtained on three different isotopically labeled ergosterol samples indicated that C-28 was derived from AdoMet, H-24 migrated to C-25 and the C-25 hydrogen on (27-)-methyl 13C-labeled ergosterol was introduced from the Re-face to produce the 25R-stereochemistry.
Yvonne Lange - One of the best experts on this subject based on the ideXlab platform.
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cholesterol movement from plasma membrane to rough endoplasmic reticulum inhibition by progesterone
Journal of Biological Chemistry, 1994Co-Authors: Yvonne LangeAbstract:Abstract The effect of progesterone on the movement of sterols from the cell surface to the rough endoplasmic reticulum (ER) was examined in rat hepatoma cells. Plasma membranes were labeled exogenously with [3H]cholesterol or [3H]Zymosterol. Translocation of the labeled sterols to the rough ER was inferred from their conversion to [3H]cholesteryl esters and [3H]cholesterol, respectively. Progesterone inhibited both of these reactions by more than 90%. The concentration for half-maximal inhibition was 0.7 microgram/ml. Progesterone did not inhibit acyl-CoA:cholesterol acyltransferase activity itself, since the steroid had no effect on the esterification of [3H]cholesterol synthesized in vitro in the rough ER from [3H]Zymosterol. Moreover, the small amount of [3H]cholesterol synthesized from plasma membrane [3H]Zymosterol in progesterone-treated intact cells was esterified at the same fractional rate as cholesterol in control cells. Subcellular fractionation of cells pulse-labeled with [3H]cholesterol and treated with progesterone suggested that the block in plasma membrane cholesterol transfer to the rough ER occurred at the level of the plasma membrane.
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Movement of Zymosterol, a precursor of cholesterol, among three membranes in human fibroblasts.
The Journal of biological chemistry, 1991Co-Authors: Yvonne Lange, F Echevarria, Theodore L. SteckAbstract:Abstract Where examined, cholesterol is synthesized in the endoplasmic reticulum; however, its precursor, Zymosterol, is found mostly in the plasma membrane. The novel implication of these disparate findings is that Zymosterol circulates within the cell. In tracing its movements, we have now established the following: (a) in human fibroblasts, Zymosterol is converted to cholesterol solely in the rough ER. (b) Little or no Zymosterol or cholesterol accumulates in the rough ER in vivo. (c) Newly synthesized Zymosterol moves to the plasma membrane without a detectable lag and with a half-time of 9 min, about twice as fast as cholesterol. (d) The pool of radiolabeled Zymosterol in the plasma membrane turns over rapidly, faster than does intracellular cholesterol. Thus, plasma membrane Zymosterol is not stagnant. (e) [3H]Zymosterol pulsed into intact cells is initially found in the plasma membrane. It is rapidly internalized and is then converted to [3H] cholesterol. Half of the [3H]cholesterol produced returns to the plasma membrane within 30 min of the initial [3H]Zymosterol pulse. (f) Nascent Zymosterol accumulates in a buoyant sterol-rich intracellular membrane before it reaches the plasma membrane. This membrane also acquires nascent cholesterol, exogenous [3H]Zymosterol pulsed into intact cells, and [3H]cholesterol synthesized from the exogenous [3H] Zymosterol. These results suggest that at least one sterol moves rapidly and in both directions among the rough endoplasmic reticulum, a sterol-rich intracellular membrane bearing nascent cholesterol, and the plasma membrane.
De-an Guo - One of the best experts on this subject based on the ideXlab platform.
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Substrate-Based Inhibitors of the (S)-Adenosyl-l-Methionine:Δ24(25)- to Δ24(28)-Sterol Methyl Transferase fromSaccharomyces cerevisiae ☆ ☆☆
Archives of biochemistry and biophysics, 1997Co-Authors: W. David Nes, De-an Guo, Wen ZhouAbstract:Abstract A series of 31 side-chain-modified analogs of cholesterol, Zymosterol, lanosterol, and cycloartenol and the steroidal alkaloids solasodine and solanidine were studied as inhibitors of ( S )-adenosyl- l -methionine:Δ 24(25) sterol methyl transferase (SMT) enzyme activity from Saccharomyces cerevisiae. Two classes of sterol methylation inhibitors were tested: substrate analogs, including mechanism-based inhibitors, and transition state analogs. Several novel sterol methylation inhibitors that contained an aza, aziridine, or ammonium group in the sterol side chain were prepared and tested for the first time. The degree and kinetic pattern of methylation inhibition were found to be influenced by the position and nature of the variant functional group introduced into the side chain. The most potent inhibitors of SMT enzyme activity were transition state analog inhibitors ( K i values of 5 to 10 n m ) that mimicked the structure and conformation of the natural substrate presumed to form in the ternary complex generated in the transition state. Steroidal alkaloids were potent competitive inhibitors with K i values ranging from 2 to 30 μ m , which is about the K m app of Zymosterol, ca. 27 μ m . An isosteric analog of the natural substrate, Zymosterol, in which the 26/27-gem-dimethyl groups were joined to form a cyclopropylidene function is shown to be a potent irreversible mechanism-based inactivator of SMT enzyme activity that exhibits competitive-type inhibition, K i 48 μ m with a K inact of 1.52 min −1 . Mechanistic implications of these results provide new insights into the topology of the ternary complex involving sterol–AdoMet–enzyme.
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Stereochemical Features of C-methylations on the Path to Δ24(28)-Methylene and Δ24(28)-Ethylidene Sterols: Studies on the Recombinant Phytosterol Methyl Transferase from Arabidopsis thaliana
Tetrahedron Letters, 1997Co-Authors: Tong Yusen, De-an Guo, Brian S. Mccourt, Anil T. Mangla, Zhou Wen-xu, Mark D Jenkins, Zhou Wen, Monica Lopez, W. David NesAbstract:Using a homogenate prepared from Escherichia coli cells that express the sterol methyl transferase (SMT) gene of Arabidopsis thaliana, migration of the hydrogen atom at C-24 to C-25 from the Re-face of the double bond was demonstrated in the biosynthesis of [27-13C] 24(28)-methyleneZymosterol (fecosterol) from [27-13C]Zymosterol and the chirality of the C-25 stereocenter (25R) was found to be retained after the stereospecific conversion of [27-13C]24(28)-methyleneZymosterol to [27-13C](24(28)Z) -ethylidenecholest-8-en-3β-ol. © 1997 Elsevier Science Ltd.
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Stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation in ergosterol biosynthesis
Tetrahedron Letters, 1996Co-Authors: Wen Zhou, De-an Guo, W. David NesAbstract:[methyl-2H3]Methionine and Zymosterol, [27-13C]lanosterol, [24-2H]lanosterol and lanosterol were separately incubated with the sterol auxotroph Saccharomyces cerevisiae strain GL7. Spectral evidence (1H and 13C-NMR) obtained on three different isotopically labeled ergosterol samples indicated that C-28 was derived from AdoMet, H-24 migrated to C-25 and the C-25 hydrogen on (27-)-methyl 13C-labeled ergosterol was introduced from the Re-face to produce the 25R-stereochemistry.
