RESUMO
The activation pathway of the chloroplastic NADP-dependent malate dehydrogenase (MDH) by reduced thioredoxin has been examined using a method based on the mechanism of thiol/disulfide interchanges, i.e. the transient formation of a mixed disulfide between the target and the reductant. This disulfide can be stabilized when each of the partners is mutated in the less reactive cysteine of the disulfide/dithiol pair. As NADP-MDH has two regulatory disulfides per monomer, four different single cysteine mutants were examined, two for the C-terminal bridge and two for the N-terminal bridge. The results clearly show that the nucleophilic attack of thioredoxin on the C-terminal bridge proceeds through the formation of a disulfide with the most external Cys377. The results are less clear-cut for the N-terminal cysteines and suggest that the Cys24-Cys207 disulfide bridge previously proposed to be an intermediary step in MDH activation can form only when the C-terminal disulfide is reduced.
Assuntos
Malato Desidrogenase/metabolismo , Poaceae/enzimologia , Tiorredoxinas/metabolismo , Sítios de Ligação , Western Blotting , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Malato Desidrogenase/química , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , MutagêneseRESUMO
The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.
Assuntos
Arginina/metabolismo , Magnoliopsida/enzimologia , Malato Desidrogenase/metabolismo , Tiorredoxinas/metabolismo , Monofosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Arginina/genética , Sítios de Ligação , Catálise/efeitos dos fármacos , Dietil Pirocarbonato/farmacologia , Ativação Enzimática , Compostos de Epóxi/farmacologia , Cinética , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , Espectrometria de Massas , Mutagênese Sítio-Dirigida , NADP/química , NADP/farmacologia , Niacinamida/farmacologia , Ácido Oxaloacético/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade por SubstratoRESUMO
The chloroplastic NADP-malate dehydrogenase is activated by reduction of its N- and C-terminal disulfides by reduced thioredoxin. The activation is inhibited by NADP(+), the oxidized form of the cofactor. Previous studies suggested that the C-terminal disulfide was involved in this process. Recent structural data pointed toward a possible direct interaction between the C terminus of the oxidized enzyme and the cofactor. In the present study, the relationship between the cofactor specificity for catalysis and for inhibition of activation has been investigated by changing the cofactor specificity of the enzyme by substitution of selected residues of the cofactor-binding site. An NAD-specific thiol-regulated MDH was engineered. Its activation was inhibited by NAD(+) but no longer by NADP(+). These results demonstrate that the oxidized cofactor is bound at the same site as the reduced cofactor and support the idea of a direct interaction between the negatively charged C-terminal end of the enzyme and the positively charged nicotinamide ring of the cofactor, in agreement with the structural data. The structural requirements for cofactor specificity are modeled and discussed.
Assuntos
Cloroplastos/enzimologia , Malato Desidrogenase/metabolismo , NADP/metabolismo , NAD/metabolismo , Plantas/enzimologia , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Dissulfetos , Humanos , Cinética , Malato Desidrogenase/química , Malato Desidrogenase (NADP+) , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.
Assuntos
Escherichia coli/enzimologia , Radicais Livres/metabolismo , Nitratos/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Tirosina/análogos & derivados , Sequência de Aminoácidos , Catálise/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Dimerização , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Modelos Moleculares , Peso Molecular , Nitratos/farmacologia , Ferroproteínas não Heme/antagonistas & inibidores , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Estrutura Quaternária de Proteína/efeitos dos fármacos , Ribonucleotídeo Redutases/antagonistas & inibidores , Análise de Sequência de Proteína , Espectrofotometria , Tirosina/metabolismoRESUMO
During thioredoxin-mediated activation of chloroplastic NADP-malate dehydrogenase, a homodimeric enzyme, the interaction between subunits is known to be loosened but maintained. A modeling of the 3D structure of the protein identified Asp-101 as being potentially involved in the association between subunits through an electrostatic interaction. Indeed, upon site-directed substitution of Asp-101 by an asparagine, the mutated enzyme behaved mainly as a monomer. The mutation strongly affected the catalytical efficiency of the enzyme. The now available 3D structure of the enzyme shows that Asp-101 is protruding at the dimer interface, interacting with Arg-268 of the neighbouring subunit.
Assuntos
Ácido Aspártico/metabolismo , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Poaceae/enzimologia , Substituição de Aminoácidos/genética , Arginina/metabolismo , Ácido Aspártico/genética , Sítios de Ligação , Western Blotting , Catálise/efeitos dos fármacos , Dimerização , Dissulfetos/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Cinética , Malato Desidrogenase/genética , Malato Desidrogenase/isolamento & purificação , Malato Desidrogenase (NADP+) , Modelos Moleculares , Peso Molecular , Mutação/genética , Estrutura Quaternária de Proteína , Eletricidade Estática , Tiorredoxinas/farmacologiaRESUMO
The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.
Assuntos
Dissulfetos/metabolismo , Malato Desidrogenase/metabolismo , Folhas de Planta/enzimologia , Tiorredoxinas/metabolismo , Animais , Chlamydomonas/metabolismo , Cromatografia Líquida de Alta Pressão , Cisteína/genética , Cisteína/metabolismo , Ácido Ditionitrobenzoico/metabolismo , Ativação Enzimática , Cinética , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , Mutagênese Sítio-Dirigida/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Tiorredoxinas/genéticaRESUMO
The chloroplastic NADP malate dehydrogenase is completely inactive in its oxidized form and is activated by thiol/disulfide interchange with reduced thioredoxin. To elucidate the molecular mechanism underlying the absence of activity of the oxidized enzyme, we used site-directed mutagenesis to delete or substitute the two most C-terminal residues (C-terminal Val, penultimate Glu, both bearing negative charges). We also combined these mutations with the elimination of one or both of the possible regulatory N-terminal disulfides by mutating the corresponding cysteines. Proteins mutated at the C-terminal residues had no activity in the oxidized form but were partially inhibited when pretreated with the histidine-specific reagent diethyl pyrocarbonate before activation, showing that the active site was partially accessible. Proteins missing both N-terminal regulatory disulfides reached almost full activity without activation upon elimination of the negative charge of the penultimate Glu. These results strongly support a model where the C-terminal extension is docked into the active site through a negatively charged residue, acting as an internal inhibitor. They show also that the reduction of both N-terminal bridges is necessary to release the C-terminal extension from the active site. This is the first report for a thiol-activated enzyme of a regulatory mechanism resembling the well known intrasteric inhibition of protein kinases.
Assuntos
Grão Comestível/enzimologia , Malato Desidrogenase/antagonistas & inibidores , Substituição de Aminoácidos , Sequência de Bases , Cloroplastos/enzimologia , Primers do DNA , Dissulfetos/química , Malato Desidrogenase/química , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação ProteicaRESUMO
The malate dehydrogenase (MDH) from Streptomyces aureofaciens was purified to homogeneity and its physical and biochemical properties were studied. Its amino-terminal sequence perfectly matched the amino-terminal sequence of the MDH from Streptomyces atratus whose biochemical characteristics have never been determined. The molecular mass of the native enzyme, estimated by size-exclusion chromatography, was 70 kDa. The protein was a homodimer, with a 38-kDa subunit molecular mass. It showed a strong specificity for NADH and was much more efficient for the reduction of oxaloacetate than for the oxidation of malate, with a pH optimum of 8. Unlike MDHs from other sources, it was not inhibited by excess oxaloacetate. This first complete functional characterization of an MDH from Streptomyces shows that the enzyme is very similar in many respects to other bacterial MDHs with the notable exception of a lack of inhibition by excess substrate.
Assuntos
Malato Desidrogenase/isolamento & purificação , Streptomyces aureofaciens/enzimologia , Sequência de Aminoácidos , Cinética , Malato Desidrogenase/metabolismo , Dados de Sequência Molecular , Peso MolecularRESUMO
Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.
Assuntos
Escherichia coli/química , Prolina/química , Estrutura Secundária de Proteína , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Ativação Enzimática , Malato Desidrogenase/metabolismo , Espectrometria de Massas , Modelos Moleculares , Mutagênese Sítio-Dirigida , NADP/metabolismo , Oxirredução , Dobramento de Proteína , Relação Estrutura-Atividade , Termodinâmica , Tiorredoxinas/genéticaRESUMO
Plant NADP-dependent malate dehydrogenase is activated through thiol/disulfide interchange with reduced thioredoxin. Previous studies showed that this process involves the reduction of two different disulfides per subunit: one N-terminal, the other C-terminal. Substitution of regulatory cysteines at each end by site-directed mutagenesis and comparison of activation kinetics of the mutants led us to propose a model for the activation mechanism where the C-terminal end shielded the access to the catalytic residues, whereas the N- terminal end was involved in the slow conformational change of the active site. In the present study, we took advantage of the previous identification of the catalytic histidine residue which can be specifically derivatized by diethyl pyrocarbonate to test the accessibility of the active site. The results clearly show that in the mutants where the C-terminal bridge is open the active site histidine is freely accessible to the reagent, whereas in the mutants where the N-terminal bridge is open, the active site cannot be reached without activation, thus demonstrating the validity of the model.
Assuntos
Dissulfetos/metabolismo , Malato Desidrogenase/metabolismo , Folhas de Planta/enzimologia , Tiorredoxinas/metabolismo , Ativação Enzimática , Malato Desidrogenase/química , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The chloroplast NADP-malate dehydrogenase is activated through the reduction of two different disulfides per subunit. The activated enzyme, as well as a permanently active mutant where all four regulatory cysteines were replaced are still sensitive to thiol reagents. This observation suggested the presence of an additional important cysteine at the active site. In an attempt to identify that cysteine, site-directed mutagenesis was performed on the cDNA encoding sorghum leaf NADP-malate dehydrogenase. The replacement of Cys-175 by an alanine yielded an enzyme whose sensitivity to thiol reagents was markedly decreased whereas its catalytic activity was enhanced. This finding suggests that Cys-175 has no catalytic function but is located close to the active site.
Assuntos
Cisteína/fisiologia , Malato Desidrogenase/metabolismo , Folhas de Planta/enzimologia , Sequência de Bases , Sítios de Ligação , Ativação Enzimática , Iodoacetamida/farmacologia , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Poaceae/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Reagentes de Sulfidrila/farmacologiaRESUMO
Plant chloroplastic NADP-malate dehydrogenase is unique among malate dehydrogenases because of its reductive activation in the light and cofactor specificity. In this paper, the role of His229 in sorghum leaf protein has been investigated by site-directed mutagenesis. His229 was replaced by Asn and Gln, both mutations yielding an inactive protein. The role of a conserved Asp (Asp201) as a possible partner of His229 in catalysis has been studied by the same approach. Both Asp mutants (D201A, D201N) were only slightly active and were essentially characterized by a dramatically increased Km for oxaloacetate (45-80-fold). pH dependence of catalytic rates revealed differences between the two Asp mutants. These results demonstrate that, in sorghum leaf NADP-dependent malate dehydrogenase, His229 is involved in catalysis in interaction with Asp201.
Assuntos
Ácido Aspártico , Cloroplastos/enzimologia , Histidina , Malato Desidrogenase/metabolismo , Plantas/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/isolamento & purificação , Malato Desidrogenase (NADP+) , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A cDNA which encodes a specific member of the NADP-dependent isocitrate dehydrogenase (ICDH) multi-isoenzyme family has been isolated from a tobacco cell suspension library, and the expression pattern of ICDH transcripts examined in various plant tissues. To assign this cDNA to a specific ICDH isoenzyme, the major, cytosolic ICDH isoenzyme of tobacco leaves (ICDH1) was purified to homogeneity and its N-terminus as well as several tryptic peptides, representing 30% of the protein, were sequenced. The comparison of these amino acid sequences with the deduced protein sequence of the cDNA confirmed that this clone encodes for ICDH1. The total ICDH specific activity and protein content were higher in vascular-enriched tobacco leaf tissue than in deveined (depleted in midrib and first-order veins) leaves. Taking advantage of antibodies raised against either ICDH1 or the chloroplastic ICDH2 isoenzyme from tobacco cell suspensions, an immuno-cytochemical approach indicated that the ICDH1 isoenzyme, located in the cytosolic compartment of tobacco leaf cells, is responsible for this expression pattern. This observation was confirmed by northern blot analyses, using a specific probe obtained from the 3' non-coding region of the ICDH1 cDNA. A comparison of ICDH protein sequences shows a large degree of similarity between eukaryotes (> 60%) but a poor homology is observed when compared to Escherichia coli ICDH (< 20%). However, it was found that the amino acids implicated in substrate binding, deduced from the 3-dimensional structure of the E. coli NADP-ICDH, appear to be conserved in all the deduced eukaryotic ICDH proteins reported until now.
Assuntos
Citosol/enzimologia , Isocitrato Desidrogenase/genética , Isoenzimas/genética , Nicotiana/genética , Plantas Tóxicas , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Western Blotting , Compartimento Celular , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , Imuno-Histoquímica , Isocitrato Desidrogenase/isolamento & purificação , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Folhas de Planta/ultraestrutura , RNA Mensageiro/genética , RNA de Plantas/genética , Análise de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição Tecidual , Nicotiana/enzimologiaRESUMO
Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15,101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11,817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.
Assuntos
Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Citosol/metabolismo , Escherichia coli/genética , Tiorredoxinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Tiorredoxinas de Cloroplastos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Recombinante , Temperatura Alta , Dados de Sequência Molecular , Desnaturação Proteica , Estrutura Secundária de Proteína , Splicing de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Tiorredoxinas/química , Tiorredoxinas/isolamento & purificação , Tiorredoxinas/metabolismoRESUMO
Five different clones encoding thioredoxin homologues were isolated from Arabidopsis thaliana cDNA libraries. On the basis of the sequences they encode divergent proteins, but all belong to the cytoplasmic thioredoxins h previously described in higher plants. The five proteins obtained by overexpressing the coding sequences in Escherichia coli present typical thioredoxin activities (NADP(+)-malate dehydrogenase activation and reduction by Arabidopsis thioredoxin reductase) despite the presence of a variant active site, Trp-Cys-Pro-Pro-Cys, in three proteins in place of the canonical Trp-Cys-Gly-Pro-Cys sequence described for thioredoxins in prokaryotes and eukaryotes. Southern blots show that each cDNA is encoded by a single gene but suggest the presence of additional related sequences in the Arabidopsis genome. This very complex diversity of thioredoxins h is probably common to all higher plants, since the Arabidopsis sequences appear to have diverged very early, at the beginning of plant speciation. This diversity allows the transduction of a redox signal into multiple pathways.
Assuntos
Arabidopsis/genética , Homologia de Sequência de Aminoácidos , Tiorredoxinas/genética , Sequência de Aminoácidos , Animais , Arabidopsis/química , DNA Complementar , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Tiorredoxinas/químicaRESUMO
The cDNA fragment coding for mature chloroplast pea fructose-1,6-bisphosphatase [Fru(1,6)P2ase] was introduced by PCR into the expression vector pET-3d resulting in the construction pET-FBP. After transformation of BL21 (DE3) Escherichia coli cells by the pET-FBP plasmid and induction with isopropyl thio-beta-D-galactoside, high-level expression of the recombinant enzyme was achieved. The protein could be purified in three days by a simple procedure which includes heat treatment, ammonium sulfate fractionation, DEAE Sephacel and ACA 44 chromatographies with a yield of 20 mg/l culture. In every respect, the recombinant enzyme was similar to plant chloroplast Fru(1,6)P2ase and, in particular, its reactivity with Mg2+ and redox regulatory properties were conserved. In a second series of experiments based on three-dimensional modeling of the chloroplast protein and sequence alignments, two cysteine residues of the recombinant enzyme (Cys173 and Cys178) were mutated into serine residues. An active enzyme, which did not respond to thiol reagents and to light activation, was obtained, confirming the putative regulatory role of the insertional sequence characteristic of the chloroplast enzyme.
Assuntos
Cloroplastos/enzimologia , Frutose-Bifosfatase/genética , Regulação Enzimológica da Expressão Gênica , Genes Reguladores , Pisum sativum/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cromatografia em Gel , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Frutose-Bifosfatase/biossíntese , Frutose-Bifosfatase/química , Luz , Dados de Sequência Molecular , Mutagênese , Oligonucleotídeos/química , Plasmídeos , Proteínas RecombinantesAssuntos
Grão Comestível/enzimologia , Malato Desidrogenase/metabolismo , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dissulfetos , Ativação Enzimática/efeitos da radiação , Escherichia coli/genética , Luz , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/metabolismo , Tiorredoxina Dissulfeto Redutase/análise , Zea mays/enzimologiaRESUMO
Chloroplastic NADP(+)-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbon-fixation pathway of some C4 plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C3 plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C3-C4 photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic protein-import machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of L-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.
Assuntos
Cloroplastos/metabolismo , Cotilédone/enzimologia , Malato Desidrogenase/metabolismo , Poaceae/enzimologia , Sequência de Aminoácidos , Ácidos Carboxílicos/metabolismo , Regulação da Expressão Gênica de Plantas , Imuno-Histoquímica , Malato Desidrogenase/genética , Malato Desidrogenase (NADP+) , Dados de Sequência Molecular , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Plantas Tóxicas , Poaceae/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Amido/metabolismo , Nicotiana , Transformação GenéticaRESUMO
The flavoprotein ferredoxin-NADP reductase (FNR) was isolated from the unicellular green alga, Chlamydomonas reinhardtii. FNR is a monomeric protein containing one FAD and exhibiting ferredoxin-dependent cytochrome c reduction activity. Its complete primary structure was investigated by sequencing overlapping peptides generated by cleavage with trypsin and SV8 protease and confirmed by partial (80%) nucleotidic sequence. C. reinhardtii FNR contains 320 residues, corresponding to a calculated mass of 35,685 and 36,470 including FAD, in agreement with the values measured by laser desorption mass spectrometry. The combination of both amino acid and nucleotidic sequencing, in association with mass spectrometry of peptides, allowed the identification of two N epsilon-trimethyllysines at positions 83 and 89 and one N epsilon-dimethyllysine at position 135. Comparison of the primary structure of C. reinhardtii FNR with the known sequences shows 41-46% identity.
Assuntos
Chlamydomonas reinhardtii/genética , Ferredoxina-NADP Redutase/genética , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlamydomonas reinhardtii/enzimologia , DNA Complementar/genética , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/isolamento & purificação , Lisina/análogos & derivados , Lisina/análise , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Análise de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Thioredoxins are proteins, typically with a molecular mass of 12 kDa, that are widely, if not universally, distributed in the animal, plant, and bacterial kingdoms. Thioredoxins undergo reversible redox change through a disulfide group (S-S-->2 SH). Two cellular reductants--reduced ferredoxin and NADPH--supply the equivalents for reduction via different enzymes. The nature of the reductant serves as a basis for distinguishing and naming the two thioredoxin systems, which are discussed below in relation to their possible application in technology and medicine. Most of the discussion is referenced by general reviews. In the section dealing with animal cells, however, much of the material is quite recent. Thus, there, and elsewhere to a lesser extent, previously uncited studies are assigned specific references.