RESUMEN
The ERG6 gene from Saccharomyces cerevisiae has been functionally expressed in Escherichia coli, for the first time, yielding a protein that catalyzes the bisubstrate transfer reaction whereby the reactive methyl group from (S)-adenosyl-L-methionine is transferred stereoselectively to C-24 of the sterol side chain. The structural requirements of sterol in binding and catalysis were similar to the native protein from S. cerevisiae. Inhibition of biomethylation was observed with fecosterol and ergosterol which suggests that ergosterol may function in wild-type yeast as feedback regulator of sterol biosynthesis.
Asunto(s)
Metiltransferasas/metabolismo , Saccharomyces cerevisiae/enzimología , Esteroles/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/metabolismo , Genes Fúngicos , Cinética , Metiltransferasas/biosíntesis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , ARN Mensajero/biosíntesis , ARN Mensajero/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/enzimología , Esteroles/metabolismo , Fraccionamiento Celular , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos , Cinética , Metilación , Metiltransferasas/antagonistas & inhibidores , Microsomas/enzimología , Modelos Biológicos , Solubilidad , Especificidad por SustratoRESUMEN
Cultures of Saccharomyces cerevisiae strain GL7 (a sterol auxotroph) were incubated with nonradioactive and tritium-labeled cycloartenol, and the sterol composition of the cells was examined by chemical (GLC, TLC, HPLC, MS, 1H-NMR, and 13C-NMR) and radiotracer techniques. Several novel sterols were isolated from the cells including 14 alpha-methyl ergosta-9(11),24(28)-dien-3 beta-ol, 24 beta-methyl-9 beta,19-cyclopropyl ergost-8(14)-en-3 beta-ol, and 9 beta,19-cyclopropyl ergosta-7(8),24(28)-dien-3 beta-ol. GL7 converted [2-3H]cycloartenol to [2-3H]ergosterol in low yield (1% incorporation), whereas [2-3H]lanosterol was converted to [2-3H]ergosterol in high yield (41% incorporation). The degree of sterol absorption and transformation by GL7 was dependent on the type and amount of sterol(s) in the growth medium. The results demonstrate for the first time that yeast may transform 9 beta,19-cyclopropyl sterols to 9(11)-sterols and delta 5-sterols and that 14-demethylation of sterols may proceed in GL7 to double bond formation either in the 8(14)-position or in the 14(15)-position.
Asunto(s)
Ergosterol/metabolismo , Fitosteroles/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteroles/metabolismo , Medios de Cultivo , Cromatografía de Gases y Espectrometría de Masas , Espectrofotometría Ultravioleta , Estereoisomerismo , TriterpenosRESUMEN
Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods from Zea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-(14)C]mevalonic acid, [2-(14)C]acetate, and [2-(3)H]acetate), and stable isotopes (D2O), we discovered that hydroxymethylgutaryl CoA reductase is not "the" rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24 alpha-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an active de novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3 beta-ol. Examination of the mass spectrum and 1H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed.