RESUMO
Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of ß-xylosidases and endo-ß-1,4-xylanases in the secretomes of Aspergillus niger, by the use of chemical probes inspired by the ß-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
RESUMO
Cassava (Manihot esculenta) is a eudicotyledonous plant that produces the valine- and isoleucine-derived cyanogenic glucosides linamarin and lotaustralin with the corresponding oximes and cyanohydrins as key intermediates. CYP79 enzymes catalyzing amino acid-to-oxime conversion in cyanogenic glucoside biosynthesis are known from several plants including cassava. The enzyme system converting oxime into cyanohydrin has previously only been identified in the monocotyledonous plant great millet (Sorghum bicolor). Using this great millet CYP71E1 sequence as a query in a Basic Local Alignment Search Tool-p search, a putative functional homolog that exhibited an approximately 50% amino acid sequence identity was found in cassava. The corresponding full-length cDNA clone was obtained from a plasmid library prepared from cassava shoot tips and was assigned CYP71E7. Heterologous expression of CYP71E7 in yeast afforded microsomes converting 2-methylpropanal oxime (valine-derived oxime) and 2-methylbutanal oxime (isoleucine-derived oxime) to the corresponding cyanohydrins, which dissociate into acetone and 2-butanone, respectively, and hydrogen cyanide. The volatile ketones were detected as 2.4-dinitrophenylhydrazone derivatives by liquid chromatography-mass spectrometry. A K(S) of approximately 0.9 µm was determined for 2-methylbutanal oxime based on substrate-binding spectra. CYP71E7 exhibits low specificity for the side chain of the substrate and catalyzes the conversion of aliphatic and aromatic oximes with turnovers of approximately 21, 17, 8, and 1 min(-1) for the oximes derived from valine, isoleucine, tyrosine, and phenylalanine, respectively. A second paralog of CYP71E7 was identified by database searches and showed approximately 90% amino acid sequence identity. In tube in situ polymerase chain reaction showed that in nearly unfolded leaves, the CYP71E7 paralogs are preferentially expressed in specific cells in the endodermis and in most cells in the first cortex cell layer. In fully unfolded leaves, the expression is pronounced in the cortex cell layer just beside the epidermis and in specific cells in the vascular tissue cortex cells. Thus, the transcripts of the CYP71E7 paralogs colocalize with CYP79D1 and CYP79D2. We conclude that CYP71E7 is the oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in cassava.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosídeos/metabolismo , Manihot/enzimologia , Nitrilas/metabolismo , Oximas/metabolismo , Biocatálise , Monóxido de Carbono/metabolismo , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Regulação Enzimológica da Expressão Gênica , Glucosídeos/química , Glucosídeos/isolamento & purificação , Cinética , Manihot/genética , Nitrilas/química , Nitrilas/isolamento & purificação , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Análise Espectral , Especificidade por SubstratoRESUMO
ß-Glucosidases (BGs) from Aspergillus fumigates, Aspergillus niger, Aspergillus oryzae, Chaetomium globosum, Emericella nidulans, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. Global non-linear regression of several reactions, with or without added glucose, to a product inhibition equation enabled the concurrent derivation of the kinetic parameters k(cat), K(m), and the apparent product inhibition constant (app)K(i) for each of the enzymes. A more simple fit is not advisable to use as the determined (app)K(i) are in the same range as their K(m) for some of the tested BGs and produced glucose would in these cases interfere. The highest value for k(cat) was determined for A. fumigatus (768 s(-1)) and the lowest was a factor 9 less. K(m) varied by a factor of 3 with the lowest value determined for C. globosum (0.95 mM). The measured (app)K(i) varied a factor of 15; the hydrolytic activity of N. crassa being the most resistant to glucose with an apparent product inhibition constant of 10.1 mM. Determination of (app)K(i) using cellobiose as substrate is important as it reflects to what extent the different BGs are hydrolytically active under industrial conditions where natural substrates are hydrolyzed and the final glucose concentrations are high.
Assuntos
Celobiose/metabolismo , Celulases/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Celulases/antagonistas & inibidores , Celulases/isolamento & purificação , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/isolamento & purificação , CinéticaRESUMO
Auxin homeostasis is pivotal for normal plant growth and development. The superroot2 (sur2) mutant was initially isolated in a forward genetic screen for auxin overproducers, and SUR2 was suggested to control auxin conjugation and thereby regulate auxin homeostasis. However, the phenotype was not uniform and could not be described as a pure high auxin phenotype, indicating that knockout of CYP83B1 has multiple effects. Subsequently, SUR2 was identified as CYP83B1, a cytochrome P450 positioned at the metabolic branch point between auxin and indole glucosinolate metabolism. To investigate concomitant global alterations triggered by knockout of CYP83B1 and the countermeasures chosen by the mutant to cope with hormonal and metabolic imbalances, 10-day-old mutant seedlings were characterized with respect to their transcriptome and metabolome profiles. Here, we report a global analysis of the sur2 mutant by the use of a combined transcriptomic and metabolomic approach revealing pronounced effects on several metabolic grids including the intersection between secondary metabolism, cell wall turnover, hormone metabolism, and stress responses. Metabolic and transcriptional cross-talks in sur2 were found to be regulated by complex interactions between both positively and negatively acting transcription factors. The complex phenotype of sur2 may thus not only be assigned to elevated levels of auxin, but also to ethylene and abscisic acid responses as well as drought responses in the absence of a water deficiency. The delicate balance between these signals explains why minute changes in growth conditions may result in the non-uniform phenotype. The large phenotypic variation observed between and within the different surveys may be reconciled by the complex and intricate hormonal balances in sur2 seedlings decoded in this study.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Ácidos Indolacéticos/metabolismo , Modelos Biológicos , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plântula/genética , Plântula/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Sporopollenin is the major component of the outer pollen wall (exine). Fatty acid derivatives and phenolics are thought to be its monomeric building blocks, but the precise structure, biosynthetic route, and genetics of sporopollenin are poorly understood. Based on a phenotypic mutant screen in Arabidopsis (Arabidopsis thaliana), we identified a cytochrome P450, designated CYP704B1, as being essential for exine development. CYP704B1 is expressed in the developing anthers. Mutations in CYP704B1 result in impaired pollen walls that lack a normal exine layer and exhibit a characteristic striped surface, termed zebra phenotype. Heterologous expression of CYP704B1 in yeast cells demonstrated that it catalyzes omega-hydroxylation of long-chain fatty acids, implicating these molecules in sporopollenin synthesis. Recently, an anther-specific cytochrome P450, denoted CYP703A2, that catalyzes in-chain hydroxylation of lauric acid was also shown to be involved in sporopollenin synthesis. This shows that different classes of hydroxylated fatty acids serve as essential compounds for sporopollenin formation. The genetic relationships between CYP704B1, CYP703A2, and another exine gene, MALE STERILITY2, which encodes a fatty acyl reductase, were explored. Mutations in all three genes resulted in pollen with remarkably similar zebra phenotypes, distinct from those of other known exine mutants. The double and triple mutant combinations did not result in the appearance of novel phenotypes or enhancement of single mutant phenotypes. This implies that each of the three genes is required to provide an indispensable subset of fatty acid-derived components within the sporopollenin biosynthesis framework.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Biopolímeros/biossíntese , Carotenoides/biossíntese , Citocromo P-450 CYP4A/metabolismo , Ácidos Graxos/metabolismo , Pólen/enzimologia , Alelos , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Biocatálise , Mapeamento Cromossômico , Citocromo P-450 CYP4A/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Hidroxilação , Mutação/genética , Especificidade de Órgãos , Fenóis/metabolismo , Fenótipo , Pólen/citologia , Pólen/genética , Pólen/ultraestruturaRESUMO
CYP703 is a cytochrome P450 family specific to land plants. Typically, each plant species contains a single CYP703. Arabidopsis thaliana CYP703A2 is expressed in the anthers of developing flowers. Expression is initiated at the tetrad stage and restricted to microspores and to the tapetum cell layer. Arabidopsis CYP703A2 knockout lines showed impaired pollen development and a partial male-sterile phenotype. Scanning electron and transmission electron microscopy of pollen from the knockout plants showed impaired pollen wall development with absence of exine. The fluorescent layer around the pollen grains ascribed to the presence of phenylpropanoid units in sporopollenin was absent in the CYP703A2 knockout lines. Heterologous expression of CYP703A2 in yeast cells demonstrated that CYP703 catalyzes the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids, with a preferential hydroxylation of lauric acid at the C-7 position. Incubation of recombinant CYP703 with methanol extracts from developing flowers confirmed that lauric acid and in-chain hydroxy lauric acids are the in planta substrate and product, respectively. These data demonstrate that in-chain hydroxy lauric acids are essential building blocks in sporopollenin synthesis and enable the formation of ester and ether linkages with phenylpropanoid units. This study identifies CYP703 as a P450 family specifically involved in pollen development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Biopolímeros/biossíntese , Carotenoides/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Láuricos/metabolismo , Pólen/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catálise , Parede Celular/ultraestrutura , Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Etiquetas de Sequências Expressas , Fertilidade , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hidroxilação , Ácidos Láuricos/química , Modelos Biológicos , Mutação/genética , Filogenia , Pólen/citologia , Pólen/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.
Assuntos
Ácido Clorogênico/metabolismo , Coffea/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Coffea/enzimologia , Coffea/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Ésteres/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ácido Quínico/análogos & derivados , Ácido Quínico/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Global gene expression was analyzed in Arabidopsis (Arabidopsis thaliana) by microarrays comprising 21,500 genes. Leaf segments derived from phosphorus (P)-starved and P-replenished plants were incubated with or without sucrose (Suc) to obtain tissues with contrasting combinations of P and carbohydrate levels. Transcript profiling revealed the influence of the two factors individually and the interactions between P- and sugar-dependent gene regulation. A large number of gene transcripts changed more than 2-fold: In response to P starvation, 171 genes were induced and 16 repressed, whereas Suc incubation resulted in 337 induced and 307 repressed genes. A number of new candidate genes involved in P acquisition were discovered. In addition, several putative transcription factors and signaling proteins of P sensing were disclosed. Several genes previously identified to be sugar responsive were also regulated by P starvation and known P-responsive genes were sugar inducible. Nearly 150 genes were synergistically or antagonistically regulated by the two factors. These genes exhibit more prominent or contrasting regulation in response to Suc and P in combination than expected from the effect of the two factors individually. The genes exhibiting interactions form three main clusters with different response patterns and functionality of genes. One cluster (cluster 1) most likely represents a regulatory program to support increased growth and development when both P and carbohydrates are ample. Another cluster (cluster 3) represents genes induced to alleviate P starvation and these are further induced by carbohydrate accumulation. Thus, interactions between P and Suc reveal two different signaling programs and novel interactions in gene regulation in response to environmental factors. cis-Regulatory elements were analyzed for each factor and for interaction clusters. PHR1 binding sites were more frequent in promoters of P-regulated genes as compared to the entire Arabidopsis genome, and E2F and PHR1 binding sites were more frequent in interaction clusters 1 and 3, respectively.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genoma de Planta , Fosfatos/metabolismo , Sacarose/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular , Triticum/genética , Sequência de Aminoácidos , Sequência de Bases , Catálise , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Duplicação Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Fenóis/química , Fenóis/metabolismo , Filogenia , Poaceae/genética , Poliploidia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Triticum/enzimologia , Tiramina/análogos & derivados , Tiramina/química , Tiramina/metabolismoRESUMO
We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the omega-1 and omega-2 positions. This work was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. K((m)(app)) of 8 microM and V(max(app)) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses a microsomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed.
Assuntos
Acetatos/farmacologia , Ciclopentanos/farmacologia , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Ácidos Graxos/metabolismo , Proteínas de Plantas/química , Northern Blotting , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas , Biblioteca Gênica , Genoma de Planta , Cinética , Ácidos Láuricos/metabolismo , Microssomos/metabolismo , Modelos Químicos , Naftalenos/metabolismo , Oxilipinas , Fenobarbital/metabolismo , Ácidos Esteáricos/metabolismo , Especificidade por Substrato , Fatores de Tempo , Triticum/genéticaRESUMO
Metabolon formation and metabolic channeling in plant secondary metabolism enable plants to effectively synthesize specific natural products and to avoid metabolic interference. Channeling can involve different cell types, take advantage of compartmentalization within the same cell or proceed directly within a metabolon. New experimental approaches document the importance of channeling in the synthesis of isoprenoids, alkaloids, phenylpropanoids, flavonoids and cyanogenic glucosides. Metabolon formation and metabolic channeling in natural-product synthesis facilitate attempts to genetically engineer new pathways into plants to improve their content of valuable natural products. They also offer the opportunity to introduce new traits by genetic engineering to produce plant cultivars that adhere to the principle of substantial equivalence.
Assuntos
Complexos Multienzimáticos/metabolismo , Plantas/metabolismo , Alcaloides/biossíntese , Ativação Enzimática , Flavonoides/biossíntese , Glicosídeos/biossíntese , Isoflavonas/biossíntese , Terpenos/metabolismoRESUMO
Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.
Assuntos
Arabidopsis/genética , Nitrilas/metabolismo , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Sorghum/genética , Transcrição GênicaRESUMO
Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val. In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Glucosídeos/biossíntese , Glicosídeos/biossíntese , Lotus/metabolismo , Oxigenases de Função Mista/genética , Nitrilas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosídeos/química , Glicosídeos/química , Lotus/enzimologia , Lotus/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Nitrilas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Cytochromes P450 catalyse extremely diverse and often complex regiospecific and/or stereospecific reactions in the biosynthesis or catabolism of plant bioactive molecules. Engineered P450 expression is needed for low-cost production of antineoplastic drugs such as taxol or indole alkaloids and offers the possibility to increase the content of nutraceuticals such as phytoestrogens and antioxidants in plants. Natural products may serve important functions in plant defence and metabolic engineering of P450s is a prime target to improve plant defence against insects and pathogens. Herbicides, pollutants and other xenobiotics are metabolised by some plant P450 enzymes. These P450s are tools to modify herbicide tolerance, as selectable markers and for bioremediation.
Assuntos
Antineoplásicos/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Engenharia Genética/métodos , Herbicidas/metabolismo , Preparações Farmacêuticas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Poluentes do Solo/metabolismo , Biodegradação Ambiental , Resistência a Medicamentos/genética , Herbicidas/farmacologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Taxoides/metabolismo , Terpenos/metabolismoAssuntos
Códon , Sistema Enzimático do Citocromo P-450/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Plantas/genética , Reação em Cadeia da Polimerase/métodos , Leveduras/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Leveduras/metabolismoRESUMO
BACKGROUND: Availability of genomewide information on an increasing but still limited number of plants offers the possibility of identifying orthologues, or related genes, in species with major economical impact and complex genomes. In this paper we exploit the recently described CODEHOP primer design and PCR strategy for targeted isolation of homologues in large gene families. RESULTS: The method was tested with two different objectives. The first was to analyze the evolution of the CYP98 family of cytochrome P450 genes involved in 3-hydroxylation of phenolic compounds and lignification in a broad range of plant species. The second was to isolate an orthologue of the sorghum glucosyl transferase UGT85B1 and to determine the complexity of the UGT85 family in wheat. P450s of the CYP98 family or closely related sequences were found in all vascular plants. No related sequence was found in moss. Neither extensive duplication of the CYP98 genes nor an orthologue of UGT85B1 were found in wheat. The UGT85A subfamily was however found to be highly variable in wheat. CONCLUSIONS: Our data are in agreement with the implication of CYP98s in lignification and the evolution of 3-hydroxylation of lignin precursors with vascular plants. High conservation of the CYP98 family strongly argues in favour of an essential function in plant development. Conversely, high duplication and diversification of the UGT85A gene family in wheat suggests its involvement in adaptative response and provides a valuable pool of genes for biotechnological applications. This work demonstrates the high potential of the CODEHOP strategy for the exploration of large gene families in plants.