RESUMEN
The biochemical profile of coffee beans translates directly into quality traits, nutraceutical and health promoting properties of the coffee beverage. Ent-kaurene is the ubiquitous precursor for gibberellin biosynthesis in plants, but it also serves as an intermediate in specialized (i.e., secondary) diterpenoid metabolism that leads to a diversity of more than 1,000 different metabolites. Nutraceutical effects on human health attributed to diterpenes include antioxidant, anticarcinogenic, and anti-inflammatory properties. Cafestol (CAF) and kahweol (KAH) are two diterpenes found exclusively in the Coffea genus. Our objective was to identify and functionally characterize genes involved in the central step of ent-kaurene production. We identified 17 putative terpene synthase genes in the transcriptome of Coffea arabica. Two ent-copalyl diphosphate synthase (CaCPS) and three kaurene synthase (CaKS) were selected and manually annotated. Transcript expression profiles of CaCPS1 and CaKS3 best matched the CAF and KAH metabolite profiles in different tissues. CaCPS1 and CaKS3 proteins were heterologously expressed and functionally characterized. CaCPS1 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by CaKS3. Knowledge about the central steps of diterpene formation in coffee provides a foundation for future characterization of the subsequent enzymes involved in CAF and KAH biosynthesis.
Asunto(s)
Transferasas Alquil y Aril , Coffea , Diterpenos de Tipo Kaurano , Diterpenos , Humanos , Coffea/genética , Coffea/metabolismo , Diterpenos/química , Diterpenos de Tipo Kaurano/metabolismo , Transferasas Alquil y Aril/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Terpene synthases (TPSs) catalyze terpenoid synthesis and affect the intracellular isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) concentration. In this study, we mined the in silico genome-wide TPS genes of Hevea brasiliensis and identified 47 full-length TPS genes. They had DDXXD, DXDD, NSE/DTE, RR(X)8 W, EA(X)W, and other conserved motifs. The phylogenetic tree analysis revealed that the TPSs of H.brasiliensis (HbTPSs) were divided into five subfamilies, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g. HbTPSs were predicted to have functions in the cellular components, molecular functions, and biological processes. HbTPSs were involved in seven pathways, which were K14173, K14175, K15803, K04120, K04121, K17982, and K12742 in the secondary metabolite pathway prediction. Three-dimensional structures of HbTPSs of 7 pathways were predicted, and DDXXD, NSE/DTE, and EA(X)W conserved motifs near the binding sites were found. Cis-acting elements analysis showed that they had more cis-acting elements related to phytohormone responsiveness, which indicated that terpenoid biosynthesis might be related to phytohormone regulation. RNA-Seq analysis showed that different HbTPSs were expressed differentially in different tissues. This study's results help reveal the role of HbTPSs and their molecular mechanism and help resolve the regulatory mechanism of terpenoid biosynthesis in H.brasiliensis.
Asunto(s)
Transferasas Alquil y Aril , Hevea , Hevea/genética , Hevea/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Filogenia , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Terpenoids are a class of compounds that are found in all living organisms. In plants, some terpenoids are part of primary metabolism, but most terpenes found in plants are classified as specialized metabolites, encoded by terpene synthases (TPS). It is not obvious how to assign the putative product of a given TPS using bioinformatics tools. Phylogenetic analyses easily assign TPS into families; however members of the same TPS family can synthetize more than one terpenoid-and, in many biotechnological applications, researchers are more interested in the product of a given TPS rather than its phylogenetic profile. Automated protein annotation can be used to classify TPS based on their products, despite the family they belong to. Here, we implement an automated bioinformatics method, search_TPS, to identify TPS proteins that synthesize mono, sesqui and diterpenes in Angiosperms. We verified the applicability of the method by classifying wet lab validated TPS and applying it to find TPS proteins in Coffea arabica, C. canephora, C. eugenioides, and Quillaja saponaria. Search_TPS is a computational tool based on PERL scripts that carries out a series of HMMER searches against a curated database of TPS profile hidden Markov models. The tool is freely available at https://github.com/liliane-sntn/TPS .
Asunto(s)
Transferasas Alquil y Aril , Coffea , Transferasas Alquil y Aril/genética , Coffea/metabolismo , Biología Computacional , Humanos , Filogenia , Quillaja , Terpenos/metabolismoRESUMEN
Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and ß-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Maytenus/enzimología , Proteínas de Plantas/metabolismo , Triterpenos/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Sustitución de Aminoácidos , Vías Biosintéticas , Ciclización , Genes de Plantas , Leucina/química , Maytenus/genética , Metionina/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/biosíntesis , Triterpenos Pentacíclicos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Brasilicardinâ A (1) consists of an unusual anti/syn/anti-perhydrophenanthrene skeleton with a carbohydrate side chain and an amino acid moiety. It exhibits potent immunosuppressive activity, yet its mode of action differs from standard drugs that are currently in use. Further pre-clinical evaluation of this promising, biologically active natural product is hampered by restricted access to the ready material, as its synthesis requires both a low-yielding fermentation process using a pathogenic organism and an elaborate, multi-step total synthesis. Our semi-synthetic approach included a) the heterologous expression of the brasilicardinâ A gene cluster in different non-pathogenic bacterial strains producing brasilicardinâ A aglycone (5) in excellent yield and b) the chemical transformation of the aglycone 5 into the trifluoroacetic acid salt of brasilicardinâ A (1 a) via a short and straightforward five-steps synthetic route. Additionally, we report the first preclinical data for brasilicardinâ A.
Asunto(s)
Aminoglicósidos/metabolismo , Ingeniería Genética , Inmunosupresores/síntesis química , Transferasas Alquil y Aril/genética , Aminoglicósidos/síntesis química , Aminoglicósidos/química , Aminoglicósidos/farmacología , Animales , Productos Biológicos/síntesis química , Productos Biológicos/química , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunosupresores/química , Inmunosupresores/metabolismo , Inmunosupresores/farmacología , Ratones , Plásmidos/genética , Plásmidos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Terpenos/químicaRESUMEN
Carotenoids are plastid isoprenoid pigments that play critical roles in light harvesting, photoprotection, and phytohormone biosynthesis. They are also vitamin-A precursors and antioxidant molecules important for human nutrition. Apples (e.g. Malus x domestica Borkh), one of the most widely consumed fruits with high nutrient levels, have a very low carotenoid concentration in flesh, compared with other fruits and vegetables. This could be explained by a deficiency in carotenoid synthesis/accumulation and/or accelerated degradation. We analysed the contribution of M. domestica cv. 'Fuji' phytoene synthase (PSY) in the biosynthesis of carotenoids and determined that among four MdPSY genes present in the organism, MdPSY2 and MdPSY5 are highly expressed in leaves and during fruit ripening in line with an increment in carotenoid content in fruits. Furthermore, two representative polymorphic MdPSY2 variants were found, one with a Tyr358Phe substitution (MdPSY2_F) and the other that additionally has a six-amino-acid deletion in the signal peptide (MdPSY2_CG). MdPSY2, MdPSY5, MdPSY2_F and MdPSY2_CG are all localised in plastids. Interestingly, the polymorphic MdPSY2_F and MdPSY2_CG variants show lower enzymatic activity than the wild-type form in a heterologous complementation assay, which could be attributed to the Tyr358Phe substitution close to the active-site pocket, as was suggested by 3-D modelling analysis. The presence of polymorphic MdPSY2 variants with lower enzymatic activity could be partially responsible for the low carotenoid content in Fuji apple fruits.
Asunto(s)
Transferasas Alquil y Aril/genética , Malus/genética , Proteínas de Plantas/genética , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Secuencia de Aminoácidos , Simulación por Computador , Malus/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de SecuenciaRESUMEN
A number of antimalarial drugs interfere with the electron transport chain and heme-related reactions; however, the biosynthesis of heme derivatives in Plasmodium parasites has not been fully elucidated. Here, we characterized the steps that lead to the farnesylation of heme. After the identification of a gene encoding heme O synthase, we identified heme O synthesis in blood stage parasites through the incorporation of radioactive precursors. The presence of heme O synthesis in intraerythrocytic stages of Plasmodium falciparum was confirmed by mass spectrometry. Inabenfide and uniconazole-P appeared to interfere in heme synthesis, accordingly, parasite growth was also affected by the addition of these drugs. We conclude that heme O synthesis occurs in blood stage-P. falciparum and this pathway could be a potential target for antimalarial drugs.
Asunto(s)
Eritrocitos/parasitología , Hemo/biosíntesis , Plasmodium falciparum/metabolismo , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Antimaláricos/química , Antimaláricos/farmacología , Eritrocitos/metabolismo , Hemo/genética , Humanos , Plasmodium falciparum/genética , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Droplet digital PCR is the most robust method for absolute nucleic acid quantification. However, RNA is a very versatile molecule and its abundance is tissue-dependent. RNA quantification is dependent on a reference control to estimate the abundance. Additionally, in cancer, many cellular processes are deregulated which consequently affects the gene expression profiles. In this work, we performed microarray data mining of different childhood cancers and healthy controls. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). Gene expression was validated in 40 acute lymphoblastic leukemia samples by means of droplet digital PCR. We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. However, we observed excellent correlations among the new reference genes (p < 0.0001). We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Transferasas Alquil y Aril/genética , Proteínas Relacionadas con la Autofagia/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica/normas , Genes Esenciales/genética , Humanos , Reacción en Cadena de la Polimerasa/métodos , Complejo de la Endopetidasa Proteasomal/genética , ARN , Estándares de Referencia , TranscriptomaRESUMEN
Production of citrus, the main fruit tree crop worldwide, is severely threatened by Huanglongbing (HLB), for which as yet a cure is not available. Spread of this bacterial disease in America and Asia is intimately connected with dispersal and feeding of the insect vector Diaphorina citri, oligophagous on rutaceous host plants. Effective control of this psyllid is an important component in successful HLB management programs. Volatiles released from the non-host guava have been shown to be repellent to the psyllid and to inhibit its response to citrus odour. By analysing VOC emission from guava we identified one volatile compound, (E)-ß-caryophyllene, which at certain doses exerts a repellent effect on D. citri. Non-host plant rejection mediated by (E)-ß-caryophyllene is demonstrated here by using Arabidopsis over-expression and knock-out lines. For the first time, results indicate that genetically engineered Arabidopsis plants with modified emission of VOCs can alter the behaviour of D. citri. This study shows that transgenic plants with an inherent ability to release (E)-ß-caryophyllene can potentially be used in new protection strategies of citrus trees against HLB.
Asunto(s)
Transferasas Alquil y Aril/genética , Arabidopsis/genética , Hemípteros/efectos de los fármacos , Repelentes de Insectos/química , Plantas Modificadas Genéticamente/química , Sesquiterpenos/química , Alphaproteobacteria/patogenicidad , Animales , Arabidopsis/química , Citrus/parasitología , Hemípteros/microbiología , Repelentes de Insectos/farmacología , Insectos Vectores/efectos de los fármacos , Insectos Vectores/microbiología , Enfermedades de las Plantas/prevención & control , Sesquiterpenos Policíclicos , Psidium/química , Sesquiterpenos/farmacología , Compuestos Orgánicos Volátiles/química , Compuestos Orgánicos Volátiles/farmacologíaRESUMEN
The red seaweeds belonging to the genus Laurencia are well known as halogenated secondary metabolites producers, mainly terpenoids and acetogennins. Several of these chemicals exhibit important ecological roles and biotechnological applications. However, knowledge regarding the genes involved in the biosynthesis of these compounds is still very limited. We detected 20 different genes involved in the biosynthesis of terpenoid precursors, and 21 different genes coding for terpene synthases that are responsible for the chemical modifications of the terpenoid precursors, resulting in a high diversity of carbon chemical skeletons. In addition, we demonstrate through molecular and cytochemical approaches the occurrence of the mevalonate pathway involved in the biosynthesis of terpenes in L. dendroidea. This is the first report on terpene synthase genes in seaweeds, enabling further studies on possible heterologous biosynthesis of terpenes from L. dendroidea exhibiting ecological or biotechnological interest.
Asunto(s)
Laurencia/química , Terpenos/química , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Vías Biosintéticas , Conformación de Carbohidratos , ADN Complementario/biosíntesis , ADN Complementario/genética , Laurencia/enzimología , Laurencia/genética , Ácido Mevalónico/metabolismo , Modelos Moleculares , Terpenos/metabolismo , Transcriptoma/genéticaRESUMEN
Nicotianamine (NA) is a ubiquitous metabolite in plants that bind heavy metals, is crucial for metal homeostasis, and is also an important metal chelator that facilitates long-distance metal transport and sequestration. NA synthesis is catalyzed by the enzyme nicotianamine synthase (NAS). Eruca vesicaria subsp sativa is highly tolerant to Ni, Pb, and Zn. In this study, a gene encoding EvNAS was cloned and characterized in E. vesicaria subsp sativa. The full-length EvNAS cDNA sequence contained a 111-bp 5'-untranslated region (UTR), a 155-bp 3'-UTR, and a 966-bp open reading frame encoding 322-amino acid residues. The EvNAS genomic sequence contained no introns, which is similar to previously reported NAS genes. The deduced translation of EvNAS contained a well-conserved NAS domain (1-279 amino acids) and an LIKI-CGEAEG box identical to some Brassica NAS and to the LIRL-box in most plant NAS, which is essential for DNA binding. Phylogenetic analysis indicated that EvNAS was most closely related to Brassica rapa NAS3 within the Cruciferae, followed by Thlaspi NAS1, Camelina NAS3, and Arabidopsis NAS3. A reverse transcription-polymerase chain reaction indicated that EvNAS expression was greatest in the leaves, followed by the flower buds and hypocotyls. EvNAS was moderately expressed in the roots.
Asunto(s)
Transferasas Alquil y Aril/genética , Secuencia de Aminoácidos/genética , Brassicaceae/enzimología , Filogenia , Transferasas Alquil y Aril/biosíntesis , Clonación Molecular , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas , Hierro/metabolismo , Hojas de la Planta/genética , Raíces de PlantasRESUMEN
Pitanga (Eugenia uniflora L.) is a member of the Myrtaceae family and is of particular interest due to its medicinal properties that are attributed to specialized metabolites with known biological activities. Among these molecules, terpenoids are the most abundant in essential oils that are found in the leaves and represent compounds with potential pharmacological benefits. The terpene diversity observed in Myrtaceae is determined by the activity of different members of the terpene synthase and oxidosqualene cyclase families. Therefore, the aim of this study was to perform a de novo assembly of transcripts from E. uniflora leaves and to annotation to identify the genes potentially involved in the terpenoid biosynthesis pathway and terpene diversity. In total, 72,742 unigenes with a mean length of 1048bp were identified. Of these, 43,631 and 36,289 were annotated with the NCBI non-redundant protein and Swiss-Prot databases, respectively. The gene ontology categorized the sequences into 53 functional groups. A metabolic pathway analysis with KEGG revealed 8,625 unigenes assigned to 141 metabolic pathways and 40 unigenes predicted to be associated with the biosynthesis of terpenoids. Furthermore, we identified four putative full-length terpene synthase genes involved in sesquiterpenes and monoterpenes biosynthesis, and three putative full-length oxidosqualene cyclase genes involved in the triterpenes biosynthesis. The expression of these genes was validated in different E. uniflora tissues.
Asunto(s)
Vías Biosintéticas/genética , Genes de Plantas , Syzygium/genética , Terpenos/metabolismo , Transcriptoma/genética , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ontología de Genes , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Redes y Vías Metabólicas/genética , Anotación de Secuencia Molecular , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Syzygium/enzimologíaRESUMEN
Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants.
Asunto(s)
Transferasas Alquil y Aril/genética , Sequías , Regulación de la Expresión Génica de las Plantas , Nicotiana/fisiología , Proteínas de Plantas/genética , Solanum/genética , alfa-Tocoferol/metabolismo , Envejecimiento , Transferasas Alquil y Aril/metabolismo , Desecación , Peroxidación de Lípido , Hojas de la Planta , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Regiones Promotoras Genéticas , Solanum/metabolismo , Nicotiana/genéticaRESUMEN
Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.
Asunto(s)
Transferasas Alquil y Aril/genética , Citrus paradisi/enzimología , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Estrés Fisiológico , Ácido Abscísico/metabolismo , Transferasas Alquil y Aril/biosíntesis , Deshidratación , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Tolerancia a la Sal , Nicotiana/enzimología , Nicotiana/fisiología , Regulación hacia ArribaRESUMEN
The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSY homologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSY genes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.
Asunto(s)
Transferasas Alquil y Aril/genética , Brassica napus/enzimología , Brassica napus/genética , Secuencia de Bases , Mapeo Cromosómico , ADN de Plantas/genética , Evolución Molecular , Dosificación de Gen , Genes de Plantas , Genoma de Planta , Genotipo , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Filogenia , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species. METHODOLOGY/PRINCIPAL FINDINGS: We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5' UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system. CONCLUSIONS/SIGNIFICANCE: The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.
Asunto(s)
Transferasas Alquil y Aril/genética , Genes de Plantas/genética , Hordeum/genética , Alelos , Empalme Alternativo , Carotenoides , Expresión Génica , Geranilgeranil-Difosfato GeranilgeraniltransferasaRESUMEN
Trypanosoma cruzi, the etiologic agent for Chagas' disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Grupo Citocromo b/metabolismo , Hemo/análogos & derivados , Proteínas Protozoarias/metabolismo , Saccharomyces cerevisiae/genética , Trypanosoma cruzi/enzimología , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Grupo Citocromo b/química , Grupo Citocromo b/genética , Hemo/biosíntesis , Hemo/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Saccharomyces cerevisiae/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismoRESUMEN
D-Desosamine, or 3-(dimethylamino)-3,4,6-trideoxyglucose, is an unusual sugar found on the macrolide antibiotic erythromycin, and it has been shown to play a critical role in the biological activity of the drug. Desosamine is added to the parent aglycone via the action of a glycosyltransferase that utilizes dTDP-desosamine as its substrate. Six enzymes are required for the biosynthesis of dTDP-desosamine in Streptomyces venezuelae, with the last step catalyzed by DesVI, an N, N-dimethyltransferase. Here we describe the X-ray crystal structure determined to 2.0 A resolution of DesVI complexed with S-adenosylmethionine (SAM) and the substrate analogue UDP-benzene. Each subunit of the DesVI dimer contains a seven-stranded mixed beta-sheet flanked on either side by alpha-helices. In addition to this major tertiary structural element, there is a four-stranded antiparallel beta-sheet that provides the platform necessary for subunit-subunit assembly. On the basis of the UDP-benzene binding mode, the DesVI substrate, dTDP-3-(methylamino)-3,4,6-trideoxyglucose, has been modeled into the active site. This model places the C-6' methyl group of the sugar into a hydrophobic patch that is well-conserved among putative nucleotide-linked sugar dimethyltransferases. It is formed by Trp 140, Met 178, and Ile 200. The sugar C-2' hydroxyl sits near Tyr 14, and its C-3' amino group is properly positioned for direct in-line attack of the cofactor's reactive methyl group. While methyltransferases that catalyze single alkylations at carbons, oxygens, sulfurs, and nitrogens have been well characterized, little is known regarding enzymes capable of N,N-dimethylation reactions. As such, the ternary structure of DesVI reported here serves as a structural paradigm for a new family of dimethyltransferases that function on nucleotide-linked sugars.
Asunto(s)
Transferasas Alquil y Aril/química , Amino Azúcares/biosíntesis , Streptomyces/enzimología , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Amino Azúcares/aislamiento & purificación , Clonación Molecular , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Streptomyces/genéticaRESUMEN
The expression of mRNAs coding for 1-deoxyxylulose-5-phosphate synthase (DXS) and phytoene synthase (PSY) were studied in Dunaliella salina grown under nitrogen-sufficient (NS) and nitrogen-limited (NL) conditions. Under NS conditions growth was 2.5 times higher than under NL conditions. No differences were found in chlorophyll a content per cell, and total carotenoid content per cell was 5.33 pg 1(-1) for the NS treatment and 7.76 pg 1(-1) for the NL. DXS transcripts exhibited diminished expression under NL conditions, peaking at day 15 of cultivation in both treatments. Simultaneously, PSY transcripts exhibited constant expression under both conditions. These results suggest that these genes play an important role in the balance of photosynthetic pigments during pigment accumulation.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Carotenoides/metabolismo , Chlorophyta/genética , Transferasas/metabolismo , Transferasas Alquil y Aril/genética , Carotenoides/genética , Chlorophyta/enzimología , Regulación de la Expresión Génica/fisiología , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Nitrógeno/metabolismo , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Transferasas/genéticaRESUMEN
Isoprenoids play important roles in all living organisms as components of structural cholesterol, steroid hormones in mammals, carotenoids in plants, and ubiquinones. Significant differences occur in the length of the isoprenic side chains of ubiquinone between different organisms, suggesting that different enzymes are involved in the synthesis of these side chains. Whereas in Plasmodium falciparum the isoprenic side chains of ubiquinone contain 7-9 isoprenic units, 10-unit side chains are found in humans. In a search for the P. falciparum enzyme responsible for the biosynthesis of isoprenic side chains attached to the benzoquinone ring of ubiquinones, we cloned and expressed a putative polyprenyl synthase. Polyclonal antibodies raised against the corresponding recombinant protein confirmed the presence of the native protein in trophozoite and schizont stages of P. falciparum. The recombinant protein, as well as P. falciparum extracts, showed an octaprenyl pyrophosphate synthase activity, with the formation of a polyisoprenoid with eight isoprenic units, as detected by reverse-phase HPLC and reverse-phase TLC, and confirmed by electrospray ionization and tandem MS analysis. The recombinant and native versions of the enzyme had similar Michaelis constants with the substrates isopentenyl pyrophosphate and farnesyl pyrophosphate. The recombinant enzyme could be competitively inhibited in the presence of the terpene nerolidol. This is the first report that directly demonstrates an octaprenyl pyrophosphate synthase activity in parasitic protozoa. Given the rather low similarity of the P. falciparum enzyme to its human counterpart, decaprenyl pyrophosphate synthase, we suggest that the identified enzyme and its recombinant version could be exploited in the screening of novel drugs.