RESUMEN
14C-labelled shikimic acid and double labelled shikimic acid tritiated stereospicifically at C-6 are incorporated into 3-(3-carboxyphenyl) alanine, 3-(3-carboxy-4-hydroxyphenyl) alanine, phenylalanine, and tyrosine in Reseda lutea L., Reseda odorata L., Iris x Hollandica cv. Prof. Blauw, and Iris x hollandica cv. Wedgwood. The experiments with 14C-labelled shikimic acid confirm that the aromatic carboxyl groups and rings in 3-(3-carboxyphenyl) alanine and 3-(3-carbocy-4-hydroxyphenyl) alanine derive from the carbocyl group and ring in shikimic acid whereas the experiments with double labelled shikimic acid demonstrate that the pro-6S-hydrogen atom is retained and the pro-6R-hydrogen atom lost in the biosynthesis of 3-(3-carboxyphenyl) alanine, phenylalanine, and tyrosine in the plants used. 3H was located in the ortho-position in the aromatic rings of phenylalanine and tyrosine but in a position para to the alanine side chain of 3- (3-carboxyphenly) alanine. No 3H was found in 3- (3-carboxy-4-hydroxyphenyl) alanine. This supports a derivation of the last two compounds from chorismic acid via isochorismic acid, isoprephenic acid, and 3'-carboxyphenylpyruvic acid and 3'-carbocy-4'-hydroxyphenylphruvic acid. The 3H/14C ratio in 3-(3-carboxyphenyl) alanine was found higher than in the precursor used. This isotope effect must operate by competition between the pathways from isoprephenic acid to 3'-carbocyphenylpyruvic acid and to 3'-carbocy-4'- hydroxyphenylpyruvic acic. The proposed biosynthetic pathways for the two carboxy-substituted amino acids are in agreement with their distribution patterns in the plant kingdom and suggest that they may derive from minor changes of enzymes involved in the general pathways of aromatic biosynthesis.
Asunto(s)
Ácidos Ciclohexanocarboxílicos/metabolismo , Fenilalanina/biosíntesis , Plantas/metabolismo , Tirosina/biosíntesis , Aminoácidos/aislamiento & purificación , Benzoatos/biosíntesis , Radioisótopos de Carbono , Hidroxiácidos/biosíntesis , Modelos Químicos , Fenilalanina/análogos & derivados , Ácido Shikímico/metabolismo , Especificidad de la Especie , Tritio , Compuestos de Vinilo/metabolismoRESUMEN
There is currently intense interest in unravelling the modus operandi of type I modular polyketide synthases in order to lay the ground work for their use in the combinatorial biosynthesis of new bioactive molecules. Much of our knowledge is derived from studies on 6-deoxyerythronolide B (DEBS), the enzyme assembling the polyketide backbone of erythromycin. Work on the rifamycin polyketide synthase has revealed a number of features that differ from those seen with DEBS.
Asunto(s)
Familia de Multigenes , Rifamicinas/biosíntesis , Modelos Químicos , Complejos Multienzimáticos/metabolismoRESUMEN
BACKGROUND: The committed step in the biosynthesis of the anticancer drug taxol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this complex olefin cation cyclization cascade involving the formation of three rings and three stereogenic centers. RESULTS: Recombinant taxadiene synthase was incubated with specifically deuterated substrates, and the mechanism of cyclization was probed using MS and NMR analyses of the products to define the crucial hydrogen migration and terminating deprotonation steps. The electrophilic cyclization involves the ionization of the diphosphate with closure of the A-ring, followed by a unique intramolecular transfer of the C11 proton to the re-face of C7 to promote closure of the B/C-ring juncture, and cascade termination by proton elimination from the beta-face of C5. CONCLUSIONS: These findings provide insight into the molecular architecture of the first dedicated step of taxol biosynthesis that creates the taxane carbon skeleton, and they have broad implications for the general mechanistic capability of the large family of terpenoid cyclization enzymes.
Asunto(s)
Alquenos/metabolismo , Diterpenos/metabolismo , Isomerasas/metabolismo , Paclitaxel/biosíntesis , Plantas Medicinales , Fosfatos de Poliisoprenilo/metabolismo , Taxus/enzimología , Alquenos/química , Antineoplásicos Fitogénicos/metabolismo , Cationes , Ciclización , Deuterio , Diterpenos/química , Isomerasas/genética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación Molecular , Estructura Molecular , Paclitaxel/química , Fosfatos de Poliisoprenilo/química , Protones , Proteínas Recombinantes/metabolismo , Taxus/genéticaRESUMEN
BACKGROUND: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.
Asunto(s)
Familia de Multigenes/genética , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cósmidos , ADN Bacteriano/biosíntesis , ADN Bacteriano/genética , Glicosilación , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Naftoquinonas/aislamiento & purificación , Naftoquinonas/metabolismo , Sistemas de Lectura Abierta , Plásmidos , ARN de Transferencia/biosíntesis , ARN de Transferencia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: The ansamycin class of antibiotics are produced by various Actinomycetes. Their carbon framework arises from the polyketide pathway via a polyketide synthase (PKS) that uses an unusual starter unit. Rifamycin (rif), produced by Amycolatopsis mediterranei, is the archetype ansamycin and it is medically important. Although its basic precursors (3-amino-5-hydroxy benzoic acid AHBA, and acetic and propionic acids) had been established, and several biosynthetic intermediates had been identified, very little was known about the origin of AHBA nor had the PKS and the various genes and enzymes that modify the initial intermediate been characterized. RESULTS: A set of 34 genes clustered around the rifK gene encoding AHBA synthase were defined by sequencing all but 5 kilobases (kb) of a 95 kb contiguous region of DNA from A. mediterranei. The involvement of some of the genes in the biosynthesis of rifamycin B was examined. At least five genes were shown to be essential for the synthesis of AHBA, five genes were determined to encode the modular type I PKS that uses AHBA as the starter unit, and 20 or more genes appear to govern modification of the polyketide-derived framework, and rifamycin resistance and export. Putative regulatory genes were also identified. Disruption of the PKS genes at the end of rifA abolished rifamycin B production and resulted in the formation of P8/1-OG, a known shunt product of rifamycin biosynthesis, whereas disruption of the orf6 and orf9 genes, which may encode deoxysugar biosynthesis enzymes, had no apparent effect. CONCLUSIONS: Rifamycin production in A. mediterranei is governed by a single gene cluster consisting of structural, resistance and export, and regulatory genes. The genes characterized here could be modified to produce novel forms of the rifamycins that may be effective against rifamycin-resistant microorganisms.
Asunto(s)
Actinobacteria/química , Complejos Multienzimáticos/química , Rifamicinas/biosíntesis , Secuencia de Aminoácidos , Aminobenzoatos/metabolismo , Antibacterianos/biosíntesis , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/genética , Hidroliasas/genética , Hidroxibenzoatos , Lactamas Macrocíclicas , Datos de Secuencia Molecular , Estructura Molecular , Complejos Multienzimáticos/genética , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The nucleotide (nt) sequence of a 2326-bp BamHI-PstI DNA fragment previously isolated from Streptomyces actuosus ATCC25421 that confers resistance to the thiopeptide antibiotics, nosiheptide (Nh) and thiostrepton (Ts) upon Streptomyces lividans 1326 was determined. Two open reading frames (ORFs) were found in this 2326-bp sequence; one containing 699 nt and another of 822 nt, both reading in the same direction. The Nh-resistance gene determinant (nsh) is encoded by orf822, as determined by the 74% identity of the deduced amino acid sequence of its gene product to that of the 23S rRNA methylase encoded by the Ts-resistance gene (tsr) of Streptomyces azureus. (The respective sequences had a 72% homology.) ORF699, encoded by a gene situated upstream from orf822, contained an apparent alpha-helix-beta-turn-alpha-helix configuration which is common to DNA-binding proteins and suggests that ORF699 may be a regulatory protein. Two transcription start points (tsp) were found upstream from orf699 as demonstrated by high-resolution S1 nuclease mapping. There was also a weak tsp for the nsh gene at the first nt of ORF. Moreover, transcription was observed to read through a stem-loop structure separating the orf699 and nsh genes, as demonstrated by S1 nuclease mapping of the 3' terminus of the orf699 gene, suggesting an antitermination mechanism for regulation of nsh transcription.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Streptomyces/genética , Transcripción Genética , Secuencia de Aminoácidos , Secuencia de Bases , Codón/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores R , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Streptomyces/efectos de los fármacos , Tiazoles/farmacologíaRESUMEN
A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.
Asunto(s)
Antibacterianos/farmacología , Genes Bacterianos , Metiltransferasas/genética , Streptomyces/genética , Tioestreptona/farmacología , Secuencia de Aminoácidos , Mapeo Cromosómico , Clonación Molecular , Cósmidos , Farmacorresistencia Microbiana/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Insercional , Operón/genética , Péptido Sintasas/genética , Proteínas Ribosómicas/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The interactions between the pi cloud of the aromatic ring and the pi-electron pair of the imine double bond of aromatic oximes as model compounds of pyridoxal Schiff's bases have been studied by high-resolution carbon-13 magnetic resonance spectroscopy. The coplanarity and intramolecular hydrogen bonding have been determined by 13C-1H long range couplings. This detailed investigation of 13C-1H coupling also provides unambiguous proof of the existence of the "enol-imine" tautomers in chloroform and dimethyl sulfoxide solutions. The tautomerism between the "enol-imine" and "keto-enamine" is discussed.
Asunto(s)
Piridoxal , Bases de Schiff , Fenómenos Químicos , Química , Enlace de Hidrógeno , Modelos Químicos , Conformación MolecularRESUMEN
The natural abundance 13C magnetic resonance spectra of a series of sulfonamide drugs(sulfanilamide, sulfaguanidine,sulfathiazole, sulfasuxidine, sulfadiazine, sulfamerazine, sulfamethiazine, and sulfapyridine) have been determined at 25.15 MHz employing the pulse Fourier transform technique. The chemical shefts have been assigned with the aid of off-resonance and selective proton decoupling techniques, as well as by long-range carbon-13 proton coupling patterns.
Asunto(s)
Sulfonamidas , Fenómenos Químicos , Química , Análisis de Fourier , Espectroscopía de Resonancia Magnética , Sulfadiazina , Sulfaguanidina , Sulfamerazina , Sulfametazina , Sulfanilamidas , Sulfapiridina , SulfatiazolesRESUMEN
Nitrosourea derivatives of ergolines have been synthesized for the purpose of obtaining agents with both prolactin-and tumor-inhibitory activity. Two derivatives of 8-amino-6-methylergoline (3), 8-[3-(2-chloroethyl)-3-nitrosoureido]-1-nitroso-6-methylergoline (5c) and 8-[3-2-chloroethyl)-3-nitrosoureido]-6-methylergoline (5a), have been prepared. In addition, nitroso (7) and chloroethylcarbamyl (8) derivatives of elymoclavine (6) are reported. Compounds 5a and 5c have activity against L1210 leukemia in mice but only moderate prolactin-inhibiting activity. The chloroethylcarbamyl derivative 8 of elymoclavine is a potent prolacting inhibitor.
Asunto(s)
Antineoplásicos/síntesis química , Ergolinas/síntesis química , Animales , Ergolinas/farmacología , Leucemia L1210/tratamiento farmacológico , Ratones , Compuestos de Nitrosourea/síntesis química , Compuestos de Nitrosourea/farmacología , Prolactina/metabolismo , RatasRESUMEN
The synthesis of several N-6 derivatives of elymoclavine (3) and potential alkylating derivatives of 6-methyl-8-aminoergolines (12) is described. These compounds were screened for prolactin-inhibiting ability and 6-propyl-8-hydroxymethyl-8-ergolene (9) was found to be as active as the most potent prolactin inhibitors reported to date. The total synthesis of racemic methyl dihydrolysergate I (23), having a trans C, D ring fusion, from the tricyclic ketone 18 is also described.
Asunto(s)
Ergolinas/síntesis química , Alcaloides de Claviceps/síntesis química , Prolactina/antagonistas & inhibidores , Animales , Ergolinas/farmacología , Alcaloides de Claviceps/farmacología , Femenino , Conformación Molecular , Prolactina/sangre , Ratas , Relación Estructura-ActividadRESUMEN
A general synthetic route from elymoclavine (4a) to a variety of C-17 substituted 8-ergolenes has been established. [The C-17 position is the carbon attached to C-8 of the ergoline (1) skeleton as indicated in structure 2.] This route involves displacement reactions on the allylic chloride (4h) prepared from 4a by reaction with thionyl chloride. Conversion of the naturally occurring tricyclic clavines, chanoclavine I (5a) and isochanoclavine I (5b), to the tetracyclic clavine, agroclavine (4i), has been achieved. The new compounds prepared were tested for prolactin-inhibiting ability and were found to possess activity. One of the compounds prepared, 6-methyl-8-ergolenylacetamide (4k), was very potent, comparing favorably in activity to the best prolactin inhibitors reported to date.
Asunto(s)
Ergolinas/síntesis química , Prolactina/antagonistas & inhibidores , Animales , Ergolinas/farmacología , Masculino , Prolactina/sangre , Ratas , Relación Estructura-ActividadRESUMEN
Nitrosourea derivatives 18-22 which utilize either a piperidine or pyridine ring as a carrier group were synthesized and evaluated for anticancer activity. N'-(1-Benzyl-4-piperidinyl)-N-(2-chloroethyl)-N-nitosourea hydrogen maleate (19) exhibited good activity against intracranial L1210 leukemia as well as the mouse ependymoblastoma brain tumor system. Compound 19 exhibited comparable activity in the Lewis lung carcinoma system to N,N'-bis(2-chloroethyl)-N-nitrosourea. Replacement of the N-benzyl group in both the 3-piperidinyl- and 4-piperidinylnitrosoureas resulted in less active compounds in all tumor systems tested. The 3-pyridylnitrosourea 22 was inactive in the L-1210 leukemia system.
Asunto(s)
Antineoplásicos/síntesis química , Compuestos de Nitrosourea/síntesis química , Piperidinas/síntesis química , Piridinas/síntesis química , Animales , Leucemia Experimental/tratamiento farmacológico , Leucopenia/inducido químicamente , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Compuestos de Nitrosourea/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
The ability of four ergoline-type compounds (elymoclavine, its O-benzoate and O-carbamate, and N-methyl-6,7-secoelymoclavine) to inhibit nidation in rats was determined and found to parallel their prolactin-inhibiting activity.
Asunto(s)
Implantación del Embrión/efectos de los fármacos , Ergolinas/farmacología , Animales , Femenino , Masculino , Embarazo , RatasRESUMEN
The reaction catalyzed by bacterial GDP-mannose dehydratase (E.C. 4.2.1.47), the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose (GDP-6-deoxy-D-lyxo-hexos-4-ulose), was studied with (6R)- and (6S)-GDP-D-[4-2H1,6-3H]mannose. Conversion of these stereospecifically labeled substrates in the presence of excess unlabeled GDP-mannose into the 4-keto-6-deoxy derivatives followed by Kuhn-Roth oxidation gave acetic acid samples which were subjected to configurational analysis of the isotopically chiral methyl group. The observed F values of 64 for the material from the (6S) substrate and 31 for that from the (6R) isomer, corresponding to 48% e.e. R and 66% e.e. S configuration, respectively, of the methyl group indicate that (a) the oxidoreductase reaction involves transfer of H-4 to C-6, (b) the transfer is predominantly intramolecular, and (c) the transfer is stereospecific, H-4 replacing the C-6 hydroxyl group with inversion of configuration. A mechanism for the reaction is proposed on the basis of these results.
Asunto(s)
Guanosina Difosfato Manosa/metabolismo , Hidroliasas/metabolismo , Azúcares de Nucleósido Difosfato/metabolismo , Conformación de Carbohidratos , Cromatografía en Papel , Hidrógeno , EstereoisomerismoRESUMEN
To prepare labeled precursors for biosynthetic studies, methods for the specific introduction of tritium and deuterium into the reducing and the terminal glucose unit of maltotriose were developed. Thus [6"-3H]- and (6"-2H)-maltotriose (17) and (18) were prepared via selective methoxytritylation, deprotection and subsequent modified Pfitzner-Moffatt oxidation, followed by reduction with sodium borotritiide or sodium borodeuteride, respectively. A simple two step procedure utilizing the Lobry de Bruyn/van Ekenstein transformation gave (2-2H)maltotriose (20).
Asunto(s)
Marcaje Isotópico/métodos , Trisacáridos/síntesis química , Secuencia de Carbohidratos , Deuterio , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Tritio , beta-Amilasa/metabolismoRESUMEN
The nature of the solution conformations of the alginic acid components D-mannuronan (poly-ManA) and L-guluronan (poly-GulA) from Azotobacter vinelandii were investigated by both one- and two-dimensional n.m.r. methods. Unequivocal proton assignments for both polymers as well as their constituent monomer units were made based on chemical-shift theory, coupling constant analysis, and nuclear Overhauser enhancement measurements. These data were used to investigate the interactions of poly-GulA and poly-ManA with Ca2+ ion in aqueous medium. Based on relative crosspeak integrals measured in two-dimensional phase-sensitive NOESY spectra of free and calcium-bound polymer, a model for calcium binding is proposed.
Asunto(s)
Alginatos/química , Azotobacter vinelandii/química , Calcio/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Ácido Glucurónico , Ácidos Hexurónicos/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Ácidos Urónicos/químicaRESUMEN
Four genes, ORF 22 approximately 25, from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22 were analyzed for their involvement in the biosynthesis of the two deoxysugar moieties of the granaticins. Each gene was individually inactivated on a cosmid carrying the entire gra gene cluster and the mutant cosmids were transformed into S. coelicolor CH999. Analysis of the pattern of pigment production by the transformants revealed that each of the four ORFs is required for the formation/attachment of the L-rhodinose moiety of granaticin B, but not that of the D-olivose moiety of granaticin. Based on these results and sequence homologies a pathway of dTDP-L-rhodinose formation is proposed which implicates ORF23, and possibly also ORF 24, in the 3-deoxygenation reaction, ORF 25 in the epimerization and ORF 22 in the final 4-ketoreduction.
Asunto(s)
Monosacáridos/biosíntesis , Familia de Multigenes , Naftoquinonas/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Clonación Molecular , Cósmidos , Silenciador del Gen , Mutación , Sistemas de Lectura AbiertaRESUMEN
Five new type II manumycins, containing the hydroxyquinol mC7N unit, asukamycins A-II, B-II, C-II, D-II, E-II, were discovered in cultures of Streptomyces nodosus ssp. asukaensis. The biosynthetic origin of the type II manumycins from the type I compounds, containing an epoxyquinol mC7N unit, was deduced from the time course of production and proven by preparing [7'-13C]asukamycin A and demonstrating its incorporation into asukamycin A-II.
Asunto(s)
Antibacterianos/biosíntesis , Streptomyces/metabolismo , Antibacterianos/química , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Fermentación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Polienos/síntesis química , Polienos/química , Alcamidas Poliinsaturadas , Streptomyces/enzimologíaRESUMEN
The biogenetic origin of the angucycline antibiotics urdamycins A-D was studied by feeding experiments with isotope labeled precursors and by NMR analysis. Feeding experiments with [1-13C]acetate and [1,2-13C2]acetate show that the chromophores of urdamycins A and B and the angucycline 4-ring skeleton of the urdamycins C and D chromophores are formed from a single decapolyketide chain. The chromophores of the urdamycins C and D contain additional structural elements which derived from the amino acids tyrosine and tryptophan, respectively. The latter was shown by feeding deuterium-labeled tyrosine and 13C-labeled tryptophan derivatives. Feeding of [1-13C]glucose and of [U-13C3]glycerol proved that the C-glycosidic moiety and the three sugars (2 x L-rhodinose, 1 x D-olivose each) of the urdamycins arise from glucose. Experiments with 14C-labeled urdamycin A, obtained by biosynthesis from [14C]acetate, showed this compound to be a late precursor of the urdamycins C and D.