RESUMO
Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1-2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1-1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1-1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Mutação , Fosfatidilgliceróis/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fotossíntese/genética , Plantas Geneticamente Modificadas , Plastídeos/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Plastoquinone is a redox active lipid that serves as electron transporter in the bifunctional photosynthetic-respiratory transport chain of cyanobacteria. To examine the role of genes potentially involved in cyanobacterial plastoquinone biosynthesis, we have focused on three Synechocystis sp. PCC 6803 genes likely encoding a chorismate pyruvate-lyase (sll1797) and two 4-hydroxy-3-solanesylbenzoate decarboxylases (slr1099 and sll0936). The functions of the encoded proteins were investigated by complementation experiments with Escherichia coli mutants, by the in vitro enzyme assays with the recombinant proteins, and by the development of Synechocystis sp. single-gene knock-out mutants. Our results demonstrate that sll1797 encodes a chorismate pyruvate-lyase. In the respective knock-out mutant, plastoquinone was hardly detectable, and the mutant required 4-hydroxybenzoate for growth underlining the importance of chorismate pyruvate-lyase to initiate plastoquinone biosynthesis in cyanobacteria. The recombinant Slr1099 protein displayed decarboxylase activity and catalyzed in vitro the decarboxylation of 4-hydroxy-3-prenylbenzoate with different prenyl side chain lengths. In contrast to Slr1099, the recombinant Sll0936 protein did not show decarboxylase activity regardless of the conditions used. Inactivation of the sll0936 gene in Synechocystis sp., however, caused a drastic reduction in the plastoquinone content to levels very similar to those determined in the slr1099 knock-out mutant. This proves that not only slr1099 but also sll0936 is required for plastoquinone synthesis in the cyanobacterium. In summary, our data demonstrate that cyanobacteria produce plastoquinone exclusively via a pathway that is in the first reaction steps almost identical to ubiquinone biosynthesis in E. coli with conversion of chorismate to 4-hydroxybenzoate, which is then prenylated and decarboxylated.
Assuntos
Carboxiliases/metabolismo , Oxo-Ácido-Liases/metabolismo , Plastoquinona/metabolismo , Synechocystis/enzimologia , Carboxiliases/genética , Ácido Corísmico/química , Ácido Corísmico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Oxo-Ácido-Liases/genética , Parabenos/química , Parabenos/metabolismo , Fotossíntese/genética , Filogenia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento , Ubiquinona/metabolismoRESUMO
Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the activation of phosphatidic acid to cytidinediphosphate (CDP)-diacylglycerol, a central intermediate in glycerolipid biosynthesis in prokaryotic and eukaryotic organisms. Cytidinediphosphate-diacylglycerol is the precursor to phosphatidylinositol, phosphatidylglycerol (PG) and cardiolipin of eukaryotic phospholipids that are essential for various cellular functions. Isoforms of CDS are located in plastids, mitochondria and the endomembrane system of plants and are encoded by five genes in Arabidopsis. Two genes have previously been shown to code for the plastidial isoforms which are indispensable for the biosynthesis of plastidial PG, and thus biogenesis and function of thylakoid membranes. Here we have focused on the extraplastidial CDS isoforms, encoded by CDS1 and CDS2 which are constitutively expressed contrary to CDS3. We provide evidence that these closely related CDS genes code for membrane proteins located in the endoplasmic reticulum and possess very similar enzymatic properties. Development and analysis of Arabidopsis mutants lacking either one or both CDS1 and CDS2 genes clearly shows that these two genes have redundant functions. As reflected in the seedling lethal phenotype of the cds1cds2 double mutant, plant cells require at least one catalytically active microsomal CDS isoform for cell division and expansion. According to the altered glycerolipid composition of the double mutant in comparison with wild-type seedlings, it is likely that the drastic decrease in the level of phosphatidylinositol and the increase in phosphatidic acid cause defects in cell division and expansion.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Diacilglicerol Colinofosfotransferase/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diacilglicerol Colinofosfotransferase/genética , Diacilglicerol Colinofosfotransferase/metabolismo , Diglicerídeos , Metabolismo dos Lipídeos/genética , Mutação , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/fisiologia , Fenótipo , Fosfatidilinositóis , Plastídeos , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , TilacoidesRESUMO
Plastids are DNA-containing organelles unique to plant cells. In Arabidopsis, one-third of the genes required for embryo development encode plastid-localized proteins. To help understand the role of plastids in embryogenesis and postembryonic development, we characterized proteins of the mitochondrial transcription termination factor (mTERF) family, which in animal models, comprises DNA-binding regulators of mitochondrial transcription. Of 35 Arabidopsis mTERF proteins, 11 are plastid-localized. Genetic complementation shows that at least one plastidic mTERF, BELAYA SMERT' (BSM), is required for embryogenesis. The main postembryonic phenotypes of genetic mosaics with the bsm mutation are severe abnormalities in leaf development. Mutant bsm cells are albino, are compromised in growth, and suffer defects in global plastidic gene expression. The bsm phenotype could be phenocopied by inhibition of plastid translation with spectinomycin. Plastid translation is essential for cell viability in dicotyledonous species such as tobacco but not in monocotyledonous maize. Here, genetic interactions between BSM and the gene encoding plastid homomeric acetyl-CoA carboxylase ACC2 suggest that there is a functional redundancy in malonyl-CoA biosynthesis that permits bsm cell survival in Arabidopsis. Overall, our results indicate that biosynthesis of malonyl-CoA and plastid-derived systemic growth-promoting compounds are the processes that link plant development and plastid gene expression.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Folhas de Planta/metabolismo , Plastídeos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plastídeos/genética , Biossíntese de Proteínas/fisiologia , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismo , Zea mays/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
Erucic acid (22 : 1) is a major feedstock for the oleochemical industry. In this study, a gene stacking strategy was employed to develop transgenic Crambe abyssinica lines with increased 22 : 1 levels. Through integration of the LdLPAAT, BnFAE1 and CaFAD2-RNAi genes into the crambe genome, confirmed by Southern blot and qRT-PCR, the average levels of 18 : 1, 18 : 2 and 18 : 3 were markedly decreased and that of 22 : 1 was increased from 60% in the wild type to 73% in the best transgenic line of T4 generation. In single seeds of the same line, the 22 : 1 level could reach 76.9%, an increase of 28.0% over the wild type. The trierucin amount was positively correlated to 22 : 1 in the transgenic lines. Unlike high erucic rapeseed, the wild-type crambe contains 22 : 1 in the seed phosphatidylcholine and in the sn-2 position of triacylglycerols (5% and 8%, respectively). The transgenic line with high 22 : 1 had decreased 22 : 1 level in phosphatidylcholine, and this was negatively correlated with the 22 : 1 level at the sn-2 position of TAG. The significances of this study include (i) achieving an unprecedented level of 22 : 1 in an oil crop; (ii) disclosing mechanisms in the channelling of a triacylglycerol-specific unusual fatty acid in oil seeds; (iii) indicating potential limiting factors involved in the erucic acid biosynthesis and paving the way for further increase of this acid and (iv) development of an added value genetically modified oil crop having no risk of gene flow into feed and food crops.
Assuntos
Biotecnologia/métodos , Crambe (Planta)/metabolismo , Produtos Agrícolas/metabolismo , Ácidos Erúcicos/metabolismo , Óleos Industriais/análise , Óleos de Plantas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Brassica napus/enzimologia , Crambe (Planta)/enzimologia , Crambe (Planta)/genética , Produtos Agrícolas/enzimologia , Produtos Agrícolas/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos , Regulação da Expressão Gênica de Plantas , Hibridização Genética , Padrões de Herança/genética , Fosfatidilcolinas/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/genética , Transformação Genética , Transgenes/genética , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms. RESULTS: Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands. CONCLUSIONS: We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.
Assuntos
Aciltransferases/genética , Proteínas Aviárias/genética , Galinhas/genética , Gansos/genética , Estrigiformes/genética , Aciltransferases/química , Aciltransferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Membrana Celular/enzimologia , Galinhas/metabolismo , Glândulas Exócrinas/enzimologia , Perfilação da Expressão Gênica , Lipídeos/biossíntese , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Saccharomyces cerevisiae , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10-20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K(m) value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastoquinona/metabolismo , Bioquímica/métodos , Catálise , Membrana Celular/enzimologia , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Cromatografia em Camada Fina/métodos , Cinética , Espectrometria de Massas/métodos , Plastoquinona/química , Spinacia oleracea/metabolismo , Terpenos/químicaRESUMO
Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Processos Autotróficos/efeitos da radiação , Diacilglicerol Colinofosfotransferase/genética , Genes de Plantas/genética , Luz , Plastídeos/enzimologia , Alelos , Arabidopsis/enzimologia , Arabidopsis/ultraestrutura , Processos Autotróficos/efeitos dos fármacos , Processos Autotróficos/genética , DNA Bacteriano/genética , Diacilglicerol Colinofosfotransferase/metabolismo , Teste de Complementação Genética , Glicerofosfolipídeos/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Lipídeos de Membrana/metabolismo , Mutagênese Insercional/efeitos dos fármacos , Mutagênese Insercional/genética , Mutagênese Insercional/efeitos da radiação , Mutação/genética , Fenótipo , Plastídeos/genética , Plastídeos/efeitos da radiação , Plastídeos/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Frações Subcelulares/efeitos da radiação , Sacarose/farmacologiaRESUMO
BACKGROUND: Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. RESULTS: cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. CONCLUSION: The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.
Assuntos
Aldeído Oxirredutases/metabolismo , Proteínas Aviárias/metabolismo , Aves/metabolismo , Aldeído Oxirredutases/química , Aldeído Oxirredutases/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Aves/classificação , Aves/genética , Galinhas , Evolução Molecular , Gansos , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Estrigiformes , Especificidade por SubstratoRESUMO
The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates.
Assuntos
Cardiolipinas/química , Cardiolipinas/metabolismo , Proteínas de Transporte/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aciltransferases/metabolismo , Cardiolipinas/biossíntese , Proteínas de Transporte/genética , Diglicerídeos de Citidina Difosfato/metabolismo , Espectrometria de Massas , Fosfatidilgliceróis/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
In eukaryotes the presence of the dimeric phospholipid cardiolipin (CL) is limited to the mitochondrial membranes. It resides predominantly in the inner membrane where it interacts with components of the mitochondrial electron transfer chain. CL deficiency has previously been shown to affect abundances of the plant NADH-dehydrogenase complex and its association with dimeric cyctochrome c reductase. Using an Arabidopsis thaliana knock-out mutant for the final enzyme of CL biosynthesis we here extend current knowledge on the dependence of plant respiration on CL. By correlating respiratory enzyme abundances with enzymatic capacities in mitochondria isolated from wild type, CL deficient and CL complemented heterotrophic cell culture lines a new picture of the participation of CL in plant respiration is emerging. Data indicate a loss of a general reduction of respiratory capacity in CL deficient mitochondria which cannot solely be attributed to decreased abundances or capacities of mitochondrial electron transfer protein complexes and supercomplexes. Instead, it most likely is the result of a loss of the mobile electron carrier cytochrome c. Furthermore, enzymes of the tricarboxylic acid cycle are found to have lower maximum activities in the mutant, including the succinate dehydrogenase complex. Interestingly, abundance of the latter is not altered, indicative of a direct impact of CL deficiency on the enzymatic capacity of this electron transfer chain protein complex.
RESUMO
Photosynthetic membranes of organisms from cyanobacteria to seed plants are characterized by the neutral galactolipids and the anionic glycerolipids sulfoquinovosyldiacylglycerol and phosphatidylglycerol. Recent findings have brought new insights into the biosynthesis of the anionic membrane lipids, the evolutionary origin of the enzymes involved in this process, and the importance of phosphatidylglycerol and sulfoquinovosyldiacylgycerol in photosynthesis. Photosynthetic membranes require a defined level of anionic membrane lipids for proper function, and phosphatidylglycerol and sulfoquinovosyldiacylglycerol can substitute for each other to a certain extent. A defined level of phosphatidylglycerol is, however, indispensable for photoautotrophic growth. On the other hand, sulfoquinovosyldiacylglycerol plays a conditionally important role in enabling photosynthetic organisms to survive the phosphate-limiting conditions frequently encountered in natural habitats.
Assuntos
Glicolipídeos/biossíntese , Lipídeos de Membrana/metabolismo , Fosfatos/metabolismo , Fosfatidilgliceróis/biossíntese , Plantas/metabolismo , Proteínas de Arabidopsis/metabolismo , Hexosiltransferases/metabolismo , Fotossíntese , Plantas/genéticaRESUMO
A cDNA of Chlamydomonas reinhardtii encoding a plastidial homogentisate prenyltransferase was identified. Functional expression studies in Escherichia coli revealed that the enzyme possessed properties similar to the prenyltransferase of Arabidopsis thaliana encoded by At3g11950 but different from the phytyltransferases of A. thaliana and Synechocystis. Unlike the phytyltransferases, the C. reinhardtii and the respective A. thaliana enzyme showed highest activities with solanesyl diphosphate, but were hardly active with phytyl diphosphate. Hence, these data provide evidence that the latter represent homogentisate solanesyltransferases involved in plastoquinone-9 biosynthesis. Overexpression of At3g11950 in A. thaliana, however, suggests that the solanesyltransferase can affect tocopherol biosynthesis as well.
Assuntos
Chlamydomonas reinhardtii/genética , Dimetilaliltranstransferase/genética , Plastoquinona/metabolismo , Proteínas de Protozoários/genética , Tocoferóis/metabolismo , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Chlamydomonas reinhardtii/enzimologia , Dimetilaliltranstransferase/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Protozoários/metabolismo , Synechocystis/enzimologia , Synechocystis/genéticaRESUMO
The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Delta yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Delta expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate-diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/genética , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Cardiolipinas/biossíntese , Cátions Bivalentes/química , Diglicerídeos de Citidina Difosfato/química , Teste de Complementação Genética , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/isolamento & purificação , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/isolamento & purificaçãoRESUMO
Functional expression studies in microorganisms showed that the Arabidopsis thaliana gene At4g04870 represents the cardiolipin synthase (CLS) gene encoding a hydrophobic preprotein of 38 kDa with a cleavable signal peptide for the import into mitochondria. CLS of Arabidopsis over-expressed in Escherichia coli has an alkaline pH optimum, a strict requirement for divalent cations and a distinctly lower K(m) for cytidinediphosphate-diacylglycerol than for phosphatidylglycerol. It displayed a preference for both its substrates esterified with unsaturated acyl groups. Solubilization and purification experiments revealed that the protein requires a defined phospholipid environment, particularly the presence of cardiolipin, to acquire its catalytically active conformation.
Assuntos
Arabidopsis/enzimologia , Proteínas de Membrana/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Sequência de Bases , Western Blotting , Primers do DNARESUMO
Tocopherols, collectively known as vitamin E, are only synthesised in photosynthetic organisms. Tocopherol cyclase (TC) catalyses the formation of the chromanol headgroup of the various tocopherol isoforms. TCs from Arabidopsis and maize (Zea mays) were expressed in Escherichia coli and purified. Analysis of the enzymatic properties revealed similarities but also differences between the two enzymes. Overexpression of chimeric TC gene constructs in developing seeds of transgenic rapeseed plants enhanced and modified the relative abundance of individual tocochromanol species in the seed oil, indicating a regulatory function of the enzyme in prenyllipid metabolism.
Assuntos
Brassica napus/enzimologia , Transferases Intramoleculares/metabolismo , Sementes/enzimologia , Arabidopsis/enzimologia , Brassica napus/genética , Expressão Gênica , Genes de Plantas/genética , Concentração de Íons de Hidrogênio , Transferases Intramoleculares/genética , Transferases Intramoleculares/isolamento & purificação , Estrutura Molecular , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sementes/genética , Especificidade por Substrato , Temperatura , Tocoferóis/química , Tocoferóis/metabolismo , Zea mays/enzimologiaRESUMO
Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L(-1) resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L(-1). Based on this result, the hygromycin concentration up to 10 mg L(-1) was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first, followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimized the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 µg DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 µl enzyme was recomended for a complete digestion of up to 80 µg crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.
RESUMO
Multifunctional acyltransferases are able to catalyze the esterification of various acyl-acceptors with activated fatty acids. Here we describe the identification of four proteins from Tetrahymena thermophila that share certain properties with mammalian acyltransferases regarding their predicted transmembrane structure, their molecular mass and the typical acyltransferase motif. Expression of the Tetrahymena sequences results in production of triacylglycerols and wax esters in recombinant yeast when appropriate substrates are provided. The in vitro characterization shows, that these enzymes are capable of esterifying different acyl-acceptors including fatty alcohols, diols, diacylglycerols and isoprenols with acyl-CoA thioesters. Based on these catalytic activities and the sequence similarities of the Tetrahymena proteins with acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) family members, we conclude that we identified a new group of DGAT2-related multifunctional acyltransferases from protozoan organisms.
Assuntos
Aciltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/enzimologia , Triglicerídeos/biossíntese , Aciltransferases/química , Aciltransferases/genética , Sequência de Aminoácidos , Clonagem Molecular , Ésteres/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Tetrahymena thermophila/genética , Ceras/metabolismoRESUMO
As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.
Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Synechocystis/enzimologia , Aciltransferases/genética , Sequência de Aminoácidos , Ativação Enzimática , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato , Synechocystis/genéticaRESUMO
Genetic dissection of the lipid bilayer composition provides essential in vivo evidence for the role of individual lipid species in membrane function. To understand the in vivo role of the anionic phospholipid, phosphatidylglycerol, the loss-of-function mutation was identified and characterized in the Arabidopsis thaliana gene coding for phosphatidylglycerophosphate synthase 1, PGP1. This mutation resulted in pigment-deficient plants of the xantha type in which the biogenesis of thylakoid membranes was severely compromised. The PGP1 gene coded for a precursor polypeptide that was targeted in vivo to both plastids and mitochondria. The activity of the plastidial PGP1 isoform was essential for the biosynthesis of phosphatidylglycerol in chloroplasts, whereas the mitochondrial PGP1 isoform was redundant for the accumulation of phosphatidylglycerol and its derivative cardiolipin in plant mitochondrial membranes. Together with findings in cyanobacteria, these data demonstrated that anionic phospholipids play an important, evolutionarily conserved role in the biogenesis and function of the photosynthetic machinery. In addition, mutant analysis suggested that in higher plants, mitochondria, unlike plastids, could import phosphatidylglycerol from the endoplasmic reticulum.