Publications

• Sterol methyl transferase: enzymology and inhibition.

  Nes W.D.

  Sterol C-methylations catalyzed by the (S)-adenosyl-L-methionine: Delta(24)-sterol methyl transferase (SMT) have provided the focus for study of electrophilic alkylations, a reaction type of functional importance in C-C bond formation of natural products. SMTs occur generally in nature, but do not ... >> More

  Sterol C-methylations catalyzed by the (S)-adenosyl-L-methionine: Delta(24)-sterol methyl transferase (SMT) have provided the focus for study of electrophilic alkylations, a reaction type of functional importance in C-C bond formation of natural products. SMTs occur generally in nature, but do not occur in animal systems, suggesting that the difference in sterol synthetic pathways can be exploited therapeutically and in insect-plant interactions. The SMT genes from several plants and fungi have been cloned, sequenced and expressed in bacteria or yeast and bioengineered into tobacco or tomato plants. These enzymes share significant amino acid sequence similarity in the putative sterol and AdoMet binding sites. Investigations of the molecular recognition of sterol fitness and studies with stereospecifically labeled substrates as well as various sterol analogs assayed with native or mutant SMTs from fungi and plants have been carried out recently in our own and other laboratories. These analyses have led to an active-site model, referred to as the 'steric-electric plug' model, which is consistent with a non-covalent mechanism involving the intermediacy of a 24beta-methyl (or ethyl) sterol bound to the ternary complex. Despite the seeming differences between fungal and plant SMT activities the recent data indicate that a distinct SMT or family of SMTs exist in these organisms which bind and transform sterols according to a similar mechanistic plan. Vascular plants have been found to express different complements of C(1)/C(2)-activities in the form of at least three SMT isoforms. This enzyme multiplicity can be a target of regulatory control to affect phytosterol homeostasis in transgenic plants. The state of our current understanding of SMT enzymology and inhibition is presented. << Less

  Biochim Biophys Acta 1529:63-88(2000) [PubMed] [EuropePMC]

• Sterol C-methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition.

  Mangla A.T., Nes W.D.

  The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii, a non-photosynthetic, yeast-like alga, was found to C-methylate appropriate delta24(25)-sterol acceptor molecules to delta25(27)-24beta-methyl products stereoselectively. Incubation with pairs of substrates--[2H ... >> More

  The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii, a non-photosynthetic, yeast-like alga, was found to C-methylate appropriate delta24(25)-sterol acceptor molecules to delta25(27)-24beta-methyl products stereoselectively. Incubation with pairs of substrates--[2H3-methyl]AdoMet and cycloartenol, and AdoMet and [27-(13)C]lanosterol--followed by 1H and 13C NMR analysis of the isotopically labeled products demonstrated the si-face (beta-face attack) mechanism of C-methylation and the regiospecificity of delta25(27)-double bond formation from the pro-Z methyl group (C27) on lanosterol. The enzyme has a substrate preference for a sterol with a 3beta-hydroxyl group, a planar nucleus and a side chain oriented into a 'right-handed' structure (20R-chirality) characteristic of the native substrate, cycloartenol. The apparent native molecular weight of the SMT was determined to be approximately 154,000, as measured by Superose 6 FPLC. A series of sterol analogues which contain heteroatoms substituted for C24 and C25 or related structural modifications, including steroidal alkaloids, havs been used to probe further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modifications of a positively charged moiety in the form of an ammonium group substituted for carbon at C25, C24, C23 or C22 are particularly potent non-competitive inhibitors (Ki for the most potent inhibitor tested, 25-azacycloartanol, was ca. 2 nM, four orders of magnitude less than the Km for cycloartenol of 28 microM), supporting the intermediacy of the 24-methyl C24(25)-carbenium ion intermediate. Ergosterol, but neither cholesterol nor sitosterol, was found to inhibit SMT activity (Ki = 80 microM). The combination of results suggests that the interrelationships of substrate functional groups within the active center of a delta24(25) to delta25(27) 24beta-methyl-SMT could be approximated thereby allowing the rational design of C-methylation inhibitors to be formulated and tested. << Less

  Bioorg Med Chem 8:925-936(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase.

  Nes W.D., Janssen G.G., Bergenstrahle A.

  The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the micr ... >> More

  The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated. Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time. The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g. those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate. From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT. In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates. Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation. Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W. D., Benson, M., Lundin, R. E., and Le, P. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5759-5763). << Less

  J Biol Chem 266:15202-15212(1991) [PubMed] [EuropePMC]

• Mechanism and structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase from Saccharomyces cerevisiae.

  Venkatramesh M., Guo D.A., Jia Z., Nes W.D.

  The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase (SMT) from Saccharomyces cerevisiae strain GL7 have been probed with AdoMet, (S)-adenosyl-L-homocysteine, a series of 35 sterol substrates as acceptor molecules and a series of 10 ... >> More

  The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase (SMT) from Saccharomyces cerevisiae strain GL7 have been probed with AdoMet, (S)-adenosyl-L-homocysteine, a series of 35 sterol substrates as acceptor molecules and a series of 10 substrate and high energy intermediate (HEI) sterol analogues as inhibitors of biomethylation. The SMT was found to be selective for sterol, both regio- and stereochemically. The presence of an unhindered 24,25-bond, an equatorially-oriented polar group at C-3 (which must act as a proton acceptor) attached to a planar nucleus and a freely rotating side chain were obligatory structural features for sterol binding/catalysis; no essential requirement or significant harmful effects could be found for the introduction of an 8(9)-bond, 14 alpha-methyl or 9 beta,19-cyclopropyl group. Alternatively, methyl groups at C-4 prevented productive sterol binding to the SMT. Initial velocity, product inhibition, and dead-end experiments demonstrated a rapid-equilibrium random bi bi mechanism. Deuterium isotope effects developed from SMT assays containing mixtures of [3-3H]zymosterol with AdoMet or [methyl-2H3]AdoMet confirmed the operation of a random mechanism, kappa H/kappa D = 1.3. From this combination of results, the spatial relationship of the sterol substrate to AdoMet could be approximated and the topology of the sterol binding to the SMT thereby formulated. << Less

  Biochim Biophys Acta 1299:313-324(1996) [PubMed] [EuropePMC]

• Overexpression, purification, and stereochemical studies of the recombinant (S)-adenosyl-L-methionine: delta 24(25)- to delta 24(28)-sterol methyl transferase enzyme from Saccharomyces cerevisiae.

  Nes W.D., McCourt B.S., Zhou W.X., Ma J., Marshall J.A., Peek L.A., Brennan M.

  The ERG6 gene that encodes (S)-adenosyl-L-methionine: delta 24(25)-to delta 24(28)-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae was introduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21 (DE3) host cells under control of a T7 promoter. ... >> More

  The ERG6 gene that encodes (S)-adenosyl-L-methionine: delta 24(25)-to delta 24(28)-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae was introduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21 (DE3) host cells under control of a T7 promoter. This enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography. N-Terminal sequence analysis of the first 10 amino acids of the purified SMT protein confirmed the identity of the start triplet and expected primary structure. The enzyme exhibited a turnover number of 0.01/s and an isoelectric point of 5.95. A combination of Superose 6 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified SMT enzyme possessed a native molecular weight of 172,000 and was tetrameric. The purified SMT enzyme generated kinetics in which velocity versus substrate curves relative to zymosterol (preferred sterol acceptor molecule) and AdoMet were sigmoidal rather than hyperbolic, indicating enzyme cooperativity among the subunits. Studies on product formation using [27-13C]zymosterol and [2H3-methyl]AdoMet incubated with the pure SMT enzyme confirmed the reaction mechanism of sterol methylation to involve a 1,2-hydride shift of H-24 to C-25 from the Re-face of the original 24,25-double bond. Deduced amino acid sequence comparisons of the SMT polypeptide from S. cerevisiae with related sterol methyl transferase enzymes of plant and fungal origin indicate that there is a significant degree of similarity between these enzymes. Specifically, there is a conserved sequence (in yeast from amino acids ca. 79 to 92 which contains an YEXGWG motif; referred to as Region I) that is not present in other AdoMet-dependent methyl transferase enzymes. << Less

  Arch Biochem Biophys 353:297-311(1998) [PubMed] [EuropePMC]

• Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis.

  Ator M.A., Schmidt S.J., Adams J.L., Dolle R.E.

  The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is ... >> More

  The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed. << Less

  Biochemistry 28:9633-9640(1989) [PubMed] [EuropePMC]

• Substrate-based inhibitors of the (S)-adenosyl-L-methionine:delta24(25)- to delta24(28)-sterol methyl transferase from Saccharomyces cerevisiae.

  Nes W.D., Guo D., Zhou W.

  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:delta24(25)-sterol methyl transferase (SMT) enzyme activity from Saccharomyces cerevisia ... >> More

  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:delta24(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 (Ki values of 5 to 10 nM) 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 Ki values ranging from 2 to 30 microM, which is about the Kmapp of zymosterol, ca. 27 microM. 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, Ki 48 microM 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. << Less

  Arch Biochem Biophys 342:68-81(1997) [PubMed] [EuropePMC]

• Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis.

  Bouvier-Nave P., Husselstein T., Benveniste P.

  Two methyl transfers are involved in the biosynthesis of 24-methyl and 24-ethyl sterols, which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second to 24-methylene lophenol. About ten cDNA clones encoding S-adenosyl-L-methionine (AdoMet) s ... >> More

  Two methyl transfers are involved in the biosynthesis of 24-methyl and 24-ethyl sterols, which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second to 24-methylene lophenol. About ten cDNA clones encoding S-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SMTs) have been isolated so far from various plants. According to their deduced amino acid sequences, they were classified in two families, smtl and smt2; in addition, smt2 cDNAs were shown to encode a 24-methylene lophenol C24 methyltransferase [Bouvier-Navé, P., Husselstein, T., Desprez, T. & Benveniste, P. (1997) Eur. J. Biochem. 246, 518-529]. We now report the comparison of two cDNAs isolated from Nicotiana tabacum, Ntsmt1-1 which belongs to the first SMT cDNA family and Ntsmt2-1 which belongs to the second. Both cDNAs were expressed in the yeast null mutant erg6, deficient in SMT. Whereas erg6 is devoid of 24-alkyl sterols, erg6 Ntsmt1-1 contained a majority of 24-methylene sterols and erg6 Ntsmt2-1, a majority of 24-ethylidene sterols, indicating distinct functions for the expression products of these cDNAs. In the presence of AdoMet, delipidated microsomes from erg6 Ntsm1-1 efficiently converted cycloartenol into 24-methylene cycloartanol, but did not produce any 24-ethylidene lophenol upon incubation with 24-methylene lophenol. This demonstrates that cDNA Ntsmt1-1 (and most probably the other plant SMT cDNAs of the first family) encode(s) a cycloartenol C24 methyltransferase. In contrast, delipidated microsomes of erg6 Ntsmt2-1 were shown to methylate preferentially 24-methylene lophenol, as expected from an SMT encoded by an smt2 cDNA. In summary, among various cDNAs isolated from N. tabacum, one (Ntsmt1-1) belongs to the first family of plant SMT cDNAs according to its deduced amino acid sequence and was shown to encode a cycloartenol C24 methyltransferase, whereas another (Ntsmt2-1) belongs to the second family and was shown to encode a 24-methylene lophenol C24 methyltransferase. Meanwhile, two cDNAs were isolated from Oriza sativa and shown to belong to smtl and to smt2 families, respectively. These data disclose the coexistence, in a given plant species, of two distinct SMTs, each catalyzing one step of methylation in the sterol biosynthesis pathway. << Less

  Eur. J. Biochem. 256:88-96(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Stereochemistry of yeast delta 24-sterol methyl transferase.

  Acuna-Johnson A.P., Oehlschlager A.C., Pierce A.M., Pierce H.D. Jr., Czyzewska E.K.

  S-Adenosyl-l-methionine: delta 24-sterol methyl transferase (24-SMT) mediates introduction of the C-28 carbon of yeast sterols. It has been shown that sulfonium analogues of the presumptive cationic intermediates of the methylenation reaction are potent in vivo and in vitro inhibitors of this proc ... >> More

  S-Adenosyl-l-methionine: delta 24-sterol methyl transferase (24-SMT) mediates introduction of the C-28 carbon of yeast sterols. It has been shown that sulfonium analogues of the presumptive cationic intermediates of the methylenation reaction are potent in vivo and in vitro inhibitors of this process. In the presence of these inhibitors, cultures of yeast produced increased proportions of zymosterol, the natural substrate of the enzyme, while proportions of ergosterol and ergostatetraenol were decreased. New C27-sterol metabolites were also found. The in vivo inhibitory power of the analogues [I50 (microM)] was determined from the proportion of C-24 methylated sterols to C-24 nonmethylated sterols in treated cultures to be in the following order: 25-thiacholesterol iodide (0.07) > 24(S)-methyl-25-thiacholesteryl iodide (0.14) > 24(R)-methyl-25-thiacholesteryl iodide (0.25). Kinetic inhibition as revealed by radiolabeled S-adenosyl-l-methionine (SAM), crude enzyme and 25-thiacholesteryl iodide revealed this inhibitor to be uncompetitive with respect to zymosterol and competitive with respect to SAM. The greater inhibitory power of 24(S)-methyl-25-thiacholesteryl iodide compared to 24(R)-methyl-25-thiacholesteryl iodide suggests that methyl donation to delta 24 occurs from the si face. When considered in conjunction with Arigoni's previous work, the present results infer the methylenation mediated by yeast 24-SMT proceeds by alkylation from the si face of delta 24 followed by migration of a hydrogen from C-24 to C-25 across the re face and final loss of a hydrogen from C-28 on the re face. << Less

  Bioorg Med Chem 5:821-832(1997) [PubMed] [EuropePMC]