RESUMEN
The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids. Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired. Since the transit peptide-mature enzyme cleavage site could not be determined directly, a series of N-terminally truncated enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional protein; however, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme. In addition to the major product, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%). Isotopically sensitive branching experiments utilizing (4R)-[4-(2)H(1)]geranylgeranyl diphosphate confirmed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the enzyme to utilize 2, 7-cyclogeranylgeranyl diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure). Two potential mechanism-based inhibitors were tested with recombinant taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition.
Asunto(s)
Diterpenos/química , Isomerasas/genética , Paclitaxel/biosíntesis , Antineoplásicos/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli , Cromatografía de Gases y Espectrometría de Masas , Cinética , Estructura Molecular , Mutación , Plantas Medicinales , Fosfatos de Poliisoprenilo/metabolismo , Precursores de Proteínas/genética , Proteínas Recombinantes , Eliminación de Secuencia , Taxus/enzimología , Taxus/metabolismoRESUMEN
Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (Mentha x piperita) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in Escherichia coli. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.
Asunto(s)
Etiquetas de Secuencia Expresada , Extractos Vegetales/genética , Aceites de Plantas/metabolismo , ADN Complementario/metabolismo , Bases de Datos Factuales , Biblioteca de Genes , Mentha piperita , Modelos Químicos , Datos de Secuencia Molecular , Células Vegetales , Aceites de Plantas/química , Análisis de Secuencia de ADN , Terpenos/química , Terpenos/metabolismoRESUMEN
Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments generated from the pure proteins of 28 and 37 kDa revealed amino acid sequences that matched two cDNA clones obtained by random screening of a peppermint-oil gland cDNA library. The deduced sequences of both proteins showed some similarity to existing prenyltransferases, and both contained a plastid-targeting sequence. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, coexpression of the two together produced functional geranyl diphosphate synthase. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically active native and recombinant enzymes, thus confirming that geranyl diphosphate synthase is a heterodimer.
Asunto(s)
Liasas Intramoleculares/genética , Secuencia de Aminoácidos , Clonación Molecular , Dimerización , Liasas Intramoleculares/química , Liasas Intramoleculares/metabolismo , Lamiaceae/química , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The oxygenation pattern of the cyclic monoterpenoids of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In peppermint (Mentha x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol, whereas in spearmint, C6-allylic hydroxylation leads to (-)-trans-carveol. The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were used to design PCR primers with which a 500-bp amplicon was prepared. This nondegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequently confirmed as the C6-hydroxylase by functional expression using the baculovirus-Spodoptera system. The probe was also utilized to isolate two closely related full-length cDNA species from a peppermint oil gland cDNA library which were confirmed as the limonene-3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal membrane anchor, consistent with the microsomal location of the native forms, exhibit calculated molecular weights of 56,149 (spearmint) and about 56,560 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet very closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure-function relationships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Lamiaceae/enzimología , Lamiaceae/genética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Secuencia de Bases , Ciclohexenos , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Cartilla de ADN/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Limoneno , Oxigenasas de Función Mixta/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas de Plantas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Spodoptera , Especificidad por Sustrato , Terpenos/metabolismoRESUMEN
Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.
Asunto(s)
Antineoplásicos Fitogénicos/biosíntesis , Escherichia coli/genética , Isomerasas/genética , Paclitaxel/biosíntesis , Secuencia de Bases , Cartilla de ADN , Isomerasas/aislamiento & purificación , Isomerasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.
Asunto(s)
Hemiterpenos , Plantas Comestibles/enzimología , Monosacáridos de Poliisoprenil Fosfato/metabolismo , Transcetolasa/química , Transcetolasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Consenso , Escherichia coli/enzimología , Evolución Molecular , Cromatografía de Gases y Espectrometría de Masas , Humanos , Ácido Mevalónico , Datos de Secuencia Molecular , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Filogenia , Hojas de la Planta , Plantas Comestibles/clasificación , Monosacáridos de Poliisoprenil Fosfato/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transferasas/metabolismoRESUMEN
(E)-beta-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-beta-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (Km for Mg2+ approximately 150 microM; Km for Mn2+ approximately 7 microM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-beta-farnesene (85%), (Z)-beta-farnesene (8%), and delta-cadinene (5%) with the native C15 substrate farnesyl diphosphate (Km approximately 0.6 microM; Vrel = 100) and Mg2+ as cofactor, and (E)-beta-farnesene (98%) and (Z)-beta-farnesene (2%) with Mn2+ as cofactor (Vrel = 80). With the C10 analog, GDP, as substrate (Km = 1.5 microM; Vrel = 3 with Mg2+ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.
Asunto(s)
Feromonas/biosíntesis , Extractos Vegetales/genética , Pirofosfatasas/genética , Sesquiterpenos/metabolismo , Secuencia de Aminoácidos , Animales , Áfidos , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Mentha piperita , Datos de Secuencia Molecular , Extractos Vegetales/metabolismoRESUMEN
(-)-Abietic acid, the principal diterpenoid resin acid of the wound-induced oleoresin secreted by grand fir (Abies grandis), is synthesized by the cyclization of geranylgeranyl diphosphate to (-)-abieta-7(8),13(14)-diene, followed by sequential three-step oxidation of the C-18 methyl group of the olefin to a carboxyl function. The enzyme catalyzing the cyclization reaction, abietadiene synthase, was purified from stems of wounded grand fir saplings and was digested with trypsin. Amino acid sequence information from the resulting peptides allowed construction of degenerate oligonucleotide primers, which amplified a 551-base pair fragment from a wound-induced stem cDNA library. This hybridization probe was then utilized to screen the wound-induced stem cDNA library, from which three cDNA clones were isolated that were functionally expressed in Escherichia coli, thereby confirming that a single protein catalyzes the complex, multistep cyclization of geranylgeranyl diphosphate to abietadiene. cDNA isolate Ac22.1, which yielded the highest expressed level of cyclase activity, was 2861 base pairs in length and encoded an 868-amino acid open reading frame that included a putative plastidial transit peptide. Deduced amino acid sequence comparison to other terpene cyclases revealed an amino-terminal region of the abietadiene synthase, which resembles those of enzymes that employ substrate double bond protonation to initiate the carbocationic reaction cascade, and a carboxyl-terminal region of the synthase, which resembles those of enzymes that employ ionization of the substrate allylic diphosphate ester function to initiate the cyclization reaction. This apparent fusion of segments of the two distinct terpenoid cyclase types is consistent with the novel mechanism of the bifunctional abietadiene synthase in catalyzing both protonation-initiated and ionization-initiated cyclization steps.
Asunto(s)
Abietanos , Diterpenos/metabolismo , Isomerasas/genética , Fenantrenos/metabolismo , Árboles/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Biblioteca de Genes , Isomerasas/aislamiento & purificación , Isomerasas/metabolismo , Datos de Secuencia Molecular , Fosfatos de Poliisoprenilo/metabolismo , ARN de Planta/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Terpenos/metabolismo , Árboles/enzimologíaRESUMEN
The committed step of taxol (paclitaxel) biosynthesis is catalyzed by taxa-4(5),11(12)-diene synthase, a diterpene cyclase responsible for transforming the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to the parent olefin with a taxane skeleton. To obtain the corresponding cDNA clone, a set of degenerate primers was constructed based on consensus sequences of related monoterpene, sesquiterpene, and diterpene cyclases. Two of these primers amplified a 83-base pair fragment that was cyclase-like in sequence and that was employed as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA extracted from Pacific yew (Taxus brevifolia) stems. Twelve independent clones with insert size in excess of 2 kilobase pairs were isolated and partially sequenced. One of these cDNA isolates was functionally expressed in Escherichia coli, yielding a protein that was catalytically active in converting geranylgeranyl diphosphate to a diterpene olefin that was confirmed to be taxa-4(5),11(12)-diene by combined capillary gas chromatography-mass spectrometry. The sequence specifies an open reading frame of 2586 nucleotides, and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303, compared with about 79,000 previously determined for the mature native enzyme. Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes; the taxadiene synthase most closely resembles (46% identity, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir.
Asunto(s)
Isomerasas/biosíntesis , Paclitaxel/biosíntesis , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Cartilla de ADN , ADN Complementario , Escherichia coli , Cromatografía de Gases y Espectrometría de Masas , Expresión Génica , Isomerasas/química , Isomerasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , ÁrbolesRESUMEN
The oxygenation pattern of the monoterpenoids of mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-limonene. In peppermint, C3-allylic hydroxylation leads to (-)-trans-isopiperitenol that ultimately is converted to (-)-menthol, whereas in spearmint, C6-allylic hydroxylation leads to (-)-trans-carveol that is oxidized to (-)-carvone. The limonene-6-hydroxylase and the cytochrome P450 reductase were purified from the oil glands of spearmint, and the system was reconstituted. Amino acid sequences from the purified hydroxylase were utilized to design primers with which a large, non-degenerate PCR product was prepared. This probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated. This clone provides the tool for isolating the homologous cDNA species from peppermint and related Mentha species.