RESUMO
Three new peptides: (pGlu)L-ethyl, (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl, were identified in the search for pyroglutamyl oligopeptide ethyl esters in sake. The ethyl esterified peptides in sake were quantitated using stable isotope dilution analysis and additional quantitation of (pGlu)L was performed using an external standard method. The concentrations of (pGlu)L-ethyl and (pGlu)L in 33 commercial sake samples ranged from 0.16 to 1.57 mg/L and 1.49 to 7.55 mg/L, respectively. The sensory properties of the pyroglutamyl oligopeptide ethyl esters and corresponding non-esterified peptides were examined: the estimated difference threshold of (pGlu)L (2.0 mg/L) and (pGlu)L-ethyl (0.267 mg/L) was exceeded in 32 and 26 samples, respectively. Estimated thresholds of (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl were often lower than the levels in quantitated sake samples. The sensory effects of these pyroglutamyl dipeptides on a model sake quality may be negative because of their unpleasant taste, however, (pGlu)LFNP-ethyl may be positive because of its mild taste.
Assuntos
Bebidas Alcoólicas/análise , Oligopeptídeos/análise , Ácido Pirrolidonocarboxílico/química , Cromatografia Líquida de Alta Pressão , Limite de Detecção , Ácido Pirrolidonocarboxílico/normas , Padrões de Referência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
ß-cryptoxanthin (ß-Cry), a xanthophyll, is unlike other abundant carotenoids, such as α-carotene, ß-carotene, lycopene, lutein, and zeaxanthin. It is not found in most fruits or vegetables but is found only in specific fruits, such as hot chili pepper, persimmon, and citrus fruits. Because recent reports suggest that ß-Cry intake is beneficial to human health, the xanthophyll requires further investigation. Although ß-Cry accumulates in the fruit of wild raspberry, Rubus palmatus, it is not present in cultivated raspberry. In the present study, two wild raspberry species were studied-R. palmatus, which accumulates ß-Cry in the fruit, and R. crataegifolius, which does not accumulate ß-Cry. Four carotenoid biosynthetic enzymes derived from these two species were analyzed-phytoene synthase (PSY), lycopene ß-cyclase (LCYb), ß-carotene hydroxylase (HYb), and zeaxanthin epoxidase (ZEP). Expression levels of their genes were also assessed to elucidate mechanism underlying ß-Cry accumulation. Partial gene sequences of RubPSY, RubLCYb, RubHYb, and RubZEP, isolated from immature raspberry fruits of R. palmatus, were used as probes for Northern blot analysis. RubZEP expression ceased as the fruits matured, possibly because of reduced production of zeaxanthin. ß-Cry is considered to be an intermediate compound that accumulates in the mature fruits of R. palmatus. High expression of RubPSY was detectable in the mature fruits of R. crataegifolius, and the expression of RubLCYb, RubHYb, and RubZEP was detectable during all stages of fruit maturation. In contrast, ß-Cry was absent in the mature fruits of R. crataegifolius.
Assuntos
beta-Criptoxantina/biossíntese , Carotenoides/biossíntese , Frutas/metabolismo , Complexos Multienzimáticos/metabolismo , Rubus/metabolismo , Transdução de Sinais/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Distribuição TecidualRESUMO
The diterpene pleuromutilin is a ribosome-targeting antibiotic isolated from basidiomycete fungi, such as Clitopilus pseudo-pinsitus. The functional characterization of all biosynthetic enzymes involved in pleuromutilin biosynthesis is reported and a biosynthetic pathway proposed. In vitro enzymatic reactions and mutational analysis revealed that a labdane-related diterpene synthase, Ple3, catalyzed two rounds of cyclization from geranylgeranyl diphosphate to premutilin possessing a characteristic 5-6-8-tricyclic carbon skeleton. Biotransformation experiments utilizing Aspergillus oryzae transformants possessing modification enzyme genes allowed the biosynthetic pathway from premutilin to pleuromutilin to be proposed. The present study sets the stage for the enzymatic synthesis of natural products isolated from basidiomycete fungi, which are a prolific source of structurally diverse and biologically active terpenoids.
Assuntos
Basidiomycota/genética , Diterpenos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Aspergillus oryzae/metabolismo , Basidiomycota/enzimologia , Vias Biossintéticas/genética , Ciclização , Diterpenos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Plasmídeos/genética , Plasmídeos/metabolismo , Compostos Policíclicos , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , PleuromutilinasRESUMO
Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-(14)C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B'-MTs. It was concluded that CTgSs have strict substrate specificity. The K(m) values of CTgS1 and CTgS2 were 121 and 184µM with nicotinic acid as a substrate, and 68 and 120µM with S-adenosyl-L-methionine as a substrate, respectively.
Assuntos
Alcaloides/química , Alcaloides/metabolismo , Coffea/enzimologia , Metiltransferases/química , Metiltransferases/metabolismo , Niacina/química , Niacina/metabolismo , Sequência de Aminoácidos , Catálise , Ativação Enzimática , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
The taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA). All of the high-TDA factor fractions showed a bitter or astringent taste in common, but their taste characters were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionation, using RP-HPLC and an instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of the peptides of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl ester of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably with the compounds of high hydrophobicity.
Assuntos
Bebidas Alcoólicas/análise , Ácidos Cafeicos/análise , Carvão Vegetal/química , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Ácidos Cumáricos/análise , Ésteres , Interações Hidrofóbicas e Hidrofílicas , Indóis/análise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Peptídeos/análise , PaladarRESUMO
The solanapyrone biosynthetic gene cluster was cloned from Alternaria solani. It consists of six genes-sol1-6-coding for a polyketide synthase, an O-methyltransferase, a dehydrogenase, a transcription factor, a flavin-dependent oxidase, and cytochrome P450. The prosolanapyrone synthase (PSS) encoded by sol1 was expressed in Aspergillus oryzae and its product was identified as desmethylprosolanapyrone I (8). Although PSS is closely related to the PKSs/Diels-Alderases LovB and MlcA of lovastatin and compactin biosynthesis, it did not catalyze cycloaddition. Sol5, encoding a flavin-dependent oxidase (solanapyrone synthase, SPS), was expressed in Pichia pastoris and purified. The purified recombinant SPS showed activity for the formation of (-)-solanapyrone A (1) from achiral prosolanapyrone II (2), establishing that this single enzyme catalyzes both the oxidation and the subsequent cycloaddition reaction, possibly as a Diels-Alder enzyme.
Assuntos
Alternaria/enzimologia , Proteínas Fúngicas/metabolismo , Oxirredutases Intramoleculares/metabolismo , Policetídeo Sintases/metabolismo , Biocatálise , Dicroísmo Circular , Ciclização , Proteínas Fúngicas/genética , Oxirredutases Intramoleculares/genética , Família Multigênica , Naftalenos/química , Naftalenos/metabolismo , Oxirredução , Policetídeo Sintases/genética , Pironas/química , Pironas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMO
We found renin inhibitory activity in rice. The physico-chemical data on the isolated inhibitors were identical to those of oleic acid and linoleic acid. Oleic acid and linoleic acid competitively inhibited renin activity, with K(i) values of 15.8 and 19.8 microM respectively. Other unsaturated free fatty acids also inhibited renin activity, but saturated fatty acids had no effect on it.
Assuntos
Oryza/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Renina/antagonistas & inibidores , Concentração Inibidora 50 , Ácido Linoleico/análise , Ácido Linoleico/química , Ácido Linoleico/isolamento & purificação , Ácido Linoleico/farmacologia , Ácido Oleico/análise , Ácido Oleico/química , Ácido Oleico/isolamento & purificação , Ácido Oleico/farmacologia , Inibidores de Proteases/análise , Inibidores de Proteases/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Factors affecting ferulic acid, ethyl ferulate and taste-active pyroglutamyl (pGlu) peptides levels in sake were analyzed using small-scale sake brewing tests on eighteen rice samples with differing cultivar variety, cropped year and area, and polishing rate. Ferulic acid concentration in sake was highly positively correlated with its content in rice (r = 0.782**, double asterisk indicates 1% significance level), feruloylesterase (FE) activity (r = 0.804**) and feruloylated saccharide forming activity (FSFA) (r = 0.619**) in the rice koji. The results suggested that ferulic acid in rice induced FE activity and FSFA, and these two enzymes accelerated the formation of ferulic acid in sake mash. The concentration of bitter-tasting peptides in sake was highly positively correlated with crude protein content in rice (r = 0.786**), and negatively correlated with acid carboxypeptidase (ACP) activity to (pGlu)LFGPNVNPWH (r = -0.612**), fermentation length (r = -0.820**), and pyroglutamyl leucine ((pGlu)L) concentration in sake (r = -0.502*; 5% significance level). The observation suggested that bitter-tasting peptides are initially formed in sake mash in accordance with protein content in rice, and are then hydrolyzed to smaller peptides, such as (pGlu)L. An ACP specific to the hydrolysis of bitter-tasting peptides was suggested by the observation that ACP activity to (pGlu)LFGPNVNPWH was significantly correlated (-0.612**) to their formation whereas an ACP to Cbz-Glu-Tyr was not (r = 0.220). It was suggested that (pGlu) oligo-peptide ethyl esters were formed during the decomposition of bitter-tasting peptides to which the ACP to (pGlu)LFGPNVNPWH might contribute.
Assuntos
Bebidas Alcoólicas/análise , Ácidos Cafeicos/análise , Ácidos Cumáricos/análise , Oryza/química , Peptídeos/análise , Ácidos Cafeicos/metabolismo , Ácidos Cumáricos/metabolismo , Fermentação , Hidrólise , Oryza/metabolismo , Peptídeos/metabolismo , PaladarRESUMO
The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2. Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate. The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal genomic data.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Diterpenos/análise , Diterpenos/metabolismo , Genes Fúngicos , Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/enzimologia , Clonagem Molecular , Dimetilaliltranstransferase/metabolismo , Diterpenos/química , Genoma Fúngico/genética , Espectroscopia de Ressonância Magnética , Família Multigênica , Estrutura Terciária de Proteína , Análise de Sequência de DNARESUMO
The lichen substance, 16-O-acetyl-leucotylic acid (1), was isolated from an acetone extract of Myelochroa aurulenta and found to exhibit antiproliferative activity against HL-60 human leukemia cells. This is the first report on its anti-leukemia activity (EC(50)=21 microM) which is greater than that of leucotylic acid (2) and the structurally related anti-tumor agent, betulinic acid (4).
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Ascomicetos/química , Leucemia/patologia , Líquens/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Células HL-60 , Humanos , Triterpenos/químicaRESUMO
Elucidation of enzymatic polyether formation is a long-standing controversial issue in organic chemistry. To address this intriguing issue, identifying the actual substrate for epoxidation and sequential cyclization is essential. We selected the representative polyether ionophore, lasalocid, which has been proposed to undergo no modification at the late stage of biosynthesis. Cloning and a sequence analysis revealed seven polyketide synthase (PKS) genes, epoxidase and epoxide hydrolase genes for sequential ether formation, and several putative genes for supplying ethylmalonyl-CoA. Based on bioinformatic data, we propose the lasalocid biosynthetic pathway which involves characteristic aromatic ring formation and sequential cyclic ether formation. The finding of a thioesterase domain at the C-terminal of the seventh PKS indicates that intriguing oxidative cascade cyclization would occur after cleavage of the polyketide intermediate from PKS. Based on this observation, we have recently reported the enzymatic transformation of a bisepoxide intermediate to lasalocid with the recombinant epoxide hydrolase, Lsd19.
Assuntos
Lasalocida/biossíntese , Redes e Vias Metabólicas/genética , Família Multigênica , Streptomyces/genética , Antibacterianos/biossíntese , Clonagem Molecular , Ciclização , Compostos de Epóxi , Policetídeo Sintases/genéticaRESUMO
Polyether metabolites are an important class of natural products. Although their biosynthesis, especially construction of polyether skeletons, attracted organic chemists for many years, no experimental data on the enzymatic polyether formation has been obtained. In this study, a putative epoxide hydrolase gene lsd19 found on the biosynthetic gene cluster of an ionophore polyether lasalocid was cloned and successfully overexpressed in Escherichia coli. Using the purified Lsd19, a proposed substrate, bisepoxyprelasalocid, and its synthesized analogue were successfully converted into lasalocid A and its derivative via a 6-endo-tet cyclization mode. On the other hand, treatment of the bisepoxide with trichloroacetic acid gave isolasalocid A via a 5-exo-tet cyclization mode. Therefore, the enzymatic conversion observed in this study unambiguously showed that the bisepoxyprelasalocid is an intermediate of the lasalocid biosynthesis and that Lsd19 catalyzes the sequential cyclic ether formations involving an energetically disfavored 6-endo-tet cyclization. This is the first example of the enzymatic epoxide-opening reactions leading to a polyether natural product.
Assuntos
Epóxido Hidrolases/metabolismo , Lasalocida/biossíntese , Antibacterianos/biossíntese , Clonagem Molecular , Ciclização , Epóxido Hidrolases/biossíntese , Epóxido Hidrolases/genética , Compostos de Epóxi/síntese química , Compostos de Epóxi/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Éteres/síntese química , Éteres/metabolismo , Ionóforos/metabolismo , Polímeros/síntese química , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismoRESUMO
We were able to show the predominant incorporation of a single enantiomer and intact incorporation of multiply labelled synthetic diketide precursors (and), which established the intermediacy of cyclopropanated diketide and led to our proposal for the unprecedented biological cyclopropation, via PKS (polyketide synthase) having a novel cyclopropanase domain, in the biosynthesis of FR-900848 (1).
Assuntos
Actinobacteria/enzimologia , Acil Coenzima A/metabolismo , Ciclopropanos/farmacologia , Modelos Biológicos , Nucleosídeos/biossíntese , Nucleosídeos/farmacologia , Policetídeo Sintases/metabolismo , EstereoisomerismoRESUMO
Mutilin (4) and deoxy analogues 2 and 3 are biosynthetic precursors of pleuromutilin (1) in the later stage of biosynthesis. Precursors 2 and 3 are required for studies on the oxygenation steps in biosynthesis, and were synthesized from readily available 1 via 4 by deoxygenation of the hydroxy groups. Feeding experiments with the (2)H-labeled precursors confirmed their microbial conversion into 1.
Assuntos
Agaricales/metabolismo , Antibacterianos/metabolismo , Cetonas/metabolismo , Compostos Policíclicos/metabolismo , Deutério , Diterpenos/metabolismo , PleuromutilinasRESUMO
Elucidation of the cyclization mechanism catalyzed by terpene synthases is important for the rational engineering of terpene cyclases. We developed a chemoenzymatic method for the synthesis of systematically deuterium-labeled geranylgeranyl diphosphate (GGPP), starting from site-specifically deuterium-labeled isopentenyl diphosphates (IPPs) using IPP isomerase and three prenyltransferases. We examined the cyclization mechanism of tetracyclic diterpene phomopsene with phomopsene synthase. A detailed EI-MS analysis of phomopsene labeled at various positions allowed us to propose the structures corresponding to the most intense peaks, and thus elucidate a cyclization mechanism involving double 1,2-alkyl shifts and a 1,2-hydride shift via a dolabelladien-15-yl cation. Our study demonstrated that this newly developed method is highly sensitive and provides sufficient information for a reliable assignment of the structures of fragmented ions.
Assuntos
Alquil e Aril Transferases/metabolismo , Espectrometria de Massas/métodos , Fosfatos de Poli-Isoprenil/síntese química , Terpenos/química , Ciclização , Deutério/química , Hemiterpenos/química , Compostos Organofosforados/químicaRESUMO
BACKGROUND: The rhizome of Oni-dokoro (a wild yam, Dioscorea tokoro) has extremely bitter taste and is not generally regarded edible;, however, in northern part of Japan, such as Iwate and a part of Aomori, it is used as health promoting food. To clarify the reason, we examined the biologically active compounds in the rhizome collected at Iwate and compared them from the other area in literature. METHODS: The acetonitrile extract from northern part of Japan was purified by bioassay-guided separation using antiproliferative activity to human leukemia HL-60 cell, and protodioscin (PD) was isolated and identified by instrumental analyses as the major active compound. RESULTS: PD known as a saponin with four sugar moieties, an inhibitor for platelet aggregation, and a low density lipoprotein (LPL) lowering agent, displayed strong growth inhibitory effect to HL-60. The literature search suggested that the rhizome from other area contained dioscin and other saponins with three sugar moieties as their major component. We assume that the edible and health promoting effect of the rhizome in the particular area is partially derived from these different components. CONCLUSION: We were interested in the differences of utilization in the rhizome of wild yam Dioscorea tokoro, and examined the chemical composition in the rhizome to find protodioscin as antiproliferative compound to HL-60. In the report from other area, the rhizome exhibited dioscin as the major compound. Our study indicated that the protodioscin/dioscin composition varied regionally, although the reason is still needs to be investigated.
RESUMO
Biosynthetic studies of the antifungal agent, FR-900848, were undertaken by feeding experiments with D-[U-13C,]glucose, L-[4-13C]aspartate, [5,5-2H,]dihydrouridine and [5,5-2H2]dihydrouracil. The 5"-amino-5"-deoxy-5',6'-dihydrouridine moiety was derived from ribose and aspartate. Based on the feeding experiments, a detailed biosynthetic pathway producing the aminodeoxydihydrouridine moiety of FR-900848 was proposed.
Assuntos
Antifúngicos/metabolismo , Streptomycetaceae/metabolismo , Ácido Aspártico/metabolismo , Isótopos de Carbono/metabolismo , Ciclopropanos , Deutério/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Nucleosídeos/biossíntese , Ribose/metabolismoRESUMO
Formation of ethyl ferulate (EF) and ferulic acid (FA) under sake mash conditions was studied using feruloylated oligosaccharide (FO), prepared from rice grains, as the substrate for rice koji enzyme. EF and FA were produced from FO over six times faster than from alkyl ferulates however, under the same ethanol concentration, only small differences were observed between the EF/FA ratios when either FO or methyl ferulate were used as substrates. Esterification and hydrolysis of FO or methyl ferulate showed similar pH dependencies and similar EF/FA ratios for each substrate in all of the pH ranges tested. Ethanol concentration clearly affected the EF/FA ratio; the ratio increased as ethanol concentration increased. Formation of EF and FA in the sake mash simulated rice digest was accelerated by addition of exogenous FO. These results indicated that supply of FO to sake mash is a crucial step for EF and FA formation, and ethanol is an influencing factor in the EF/FA ratio. The rice koji enzyme reaction suggested that EF and FA are formed through a common feruloylated enzyme intermediate complex by transesterification or hydrolysis, and these reactions occur competitively.
Assuntos
Bebidas Alcoólicas , Ácidos Cafeicos/metabolismo , Ácidos Cumáricos/metabolismo , Oligossacarídeos/metabolismo , Oryza/enzimologia , Bebidas Alcoólicas/análise , Esterificação , Etanol/análise , Etanol/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Oryza/metabolismoRESUMO
1-Aminocyclopropane-l-carboxylate deaminase (ACCD) is a pyridoxal 5/-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity.
Assuntos
Carbono-Carbono Liases/metabolismo , Pyrococcus horikoshii/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Carbono-Carbono Liases/química , Clonagem Molecular , Primers do DNA , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The key step for construction of the carbon skeleton in the indole diterpenes, paxilline, and emindole DA was examined. Intact incorporation of multiply (2)H-labeled 3-geranylgeranylindole into two different fungal metabolites proves 3-geranylgeranylindole to be a biosynthetic intermediate. These results give evidence that indole diterpenes are biosynthesized via epoxidation of a common intermediate, and the subsequent cationic cyclization, analogous to those in the steroid biosynthesis. [structure: see text]