Cloning and Sequencing of a Pea cDNA Fragment Coding for Thioredoxin m

Jaramillo, J. López; Chueca, Ana; Sahrawy, Mariam; Hermoso, R.; Lázaro, Juan José; Prado, Fernando Eduardo; Gorgé, J. López

doi:10.1104/pp.105.3.1021

Cited by 11 publications

(5 citation statements)

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“…This indicates that structural elements beyond those at the active site influence the thioredoxin reactivity, too. To reveal such elements, the sequences of the employed thioredoxins~Holmgren, 1968; Jacquot et al, 1992;Lepiniec et al, 1992;Lopez-Jaramillo et al, 1994;Rivera-Madrid et al, 1995;Stein et al, 1995;Spyrou et al, 1997! were aligned.…”

Section: Resultsmentioning

confidence: 99%

“…Those for the E. coli and C. reinhardtii h proteins were available from the X-ray and NMR data~Katti et al, 1990;Mittard et al, 1997!. The 3D models of other species were constructed by homology modeling using the known sequences Holmgren, 1968;Jacquot et al, 1992;Lepiniec et al, 1992;Lopez-Jaramillo et al, 1994;Rivera-Madrid et al, 1995;Stein et al, 1995;Spyrou et al, 1997!, high degree of the sequence homology, similar spatial organization and conserved active sites and surroundings of thioredoxins. Figure 4 shows the part of the thioredoxin surface around the active site, which interacts with the target protein in the cocrystallized binary complex of the target peptide with human thioredoxin~Qin et al, 1995!.…”

Section: Resultsmentioning

confidence: 99%

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Interaction of thioredoxins with target proteins: Role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2‐oxoacid dehydrogenase complexes

et al. 1999

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The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxindependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Sitedirected mutagenesis of D26, W31, F0W12, and Y0A70~the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied! indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the a303 10 and a1 helices, the length of the a1 helix and the charges in the a2-b3 and b4-b5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes~the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11:13-28!. The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13014, 51, and 83085, which distinguish the activating and inhibiting thioredoxin disulfides.Keywords: electrostatic dipole vector; homology modeling; long-range electrostatic interactions; protein-protein recognition; structural analog; thioredoxin; 2-oxoacid dehydrogenase complex Abbreviations: E1, 2-oxoacid dehydrogenase; E1p, pyruvate dehydrogenase; E1o, 2-oxoglutarate dehydrogenase; E2, dihydrolipoate acyltransferase; E3, dihydrolipoate dehydrogenase; ThDP, thiamin diphosphate; lip, lipoate; DTNB, 5,5 9 -dithiobis-~2-nitrobenzoic acid!; NEM, N-ethylmaleimide; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; DTT, dithiothreitol; v, reaction velocity in the absence of effectors; v a , reaction velocity affected by an effector; S 0.5 , thioredoxin concentration required for the halfmaximal effect; 3D, three-dimensional.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Interaction of thioredoxins with target proteins: Role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2‐oxoacid dehydrogenase complexes

et al. 1999

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“…A cDNA coding for pea Trx m had been previously cloned into the vector pTZ18R (LÓpez-Jaramillo et al, 1994). Because of the low yield of this recombinant (T,) in the heterologous expression in E. coli, we subcloned the cDNA fragment coding for mature pea Trx m in the high expression plasmid pET-12a.…”

Section: Construction Of the Pet-trx M Recombinantmentioning

confidence: 99%

“…A similar feature has been described for FBPase and Trx m from pea (Pisum sativum L.), even in the absence of modulators (López-Jaramillo et al, 1995). To analyze this different behavior between Trx m from pea and spinach (Spinacia oleracea L.), we cloned and sequenced the Trx m from pea (López-Jaramillo et al, 1994), since the primary structure of spinach Trx m had been previously determined. We now describe a system that, by heterologous expression of the corresponding clone, provides a high yield of homogeneous recombinant pea Trx m, which is necessary for crystallographic and thermodynamic studies.…”

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confidence: 99%

High-Yield Expression of Pea Thioredoxin m and Assessment of Its Efficiency in Chloroplast Fructose-1,6-Bisphosphatase Activation

et al. 1997

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A cDNA clone encoding pea (%um sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-lZa, the recombinant supplied 20 mg Trx m/2. Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical t o the pea leaf native protein. Chloroplast FBPase (EC 3.1.3.11) is a highly regulated enzyme of the Benson-Calvin pathway (Bassham and Krause, 1969). It becomes active in the dark-light transition by reduction of an essential -S-S-bridge of the enzyme molecule, which promotes a conformational change to an active form (Jacquot, 1984). This reduction occurs via the Trx system, by which reduction equivalents from photosynthetic electron transport are channeled to Trx in a process catalyzed by the enzyme Fd-Trx reductase; the reduced Trx, in turn, transfers the reduction equivalents to FBPase by thiol-disulfide exchange (Buchanan, 1980;Scheibe, 1990 parts, both chloroplast Trxs show the same active cluster (-W-C-G-P-C-) and similar molecular size and folding (Eklund et al., 1991). However, their primary structures are quite different; the m form is closer to prokaryotic Trxs, and thef form is more similar to that from mammals (Hartman et al., 1990). Spinach Trxf is functional in the activation of FBPase and other chloroplast enzymes, whereas Trx m has been described as specific in NADP-malate dehydrogenase activation (Jacquot et al., 1978;Schiirmann et al., 1981). However, in the presence of the modulators Ca2' and Fru-1,6-bisphosphate, FBPase is also activated by Trx m (Schiirmann et al., 1985). A similar feature has been de- MATERIALS AND METHODS Chemicals and Biological MaterialRestriction enzymes, PCR reagents, isopropyl-P-Dthiogalactoside, substrates, and auxiliary enzymes for determination of FBPase activity were provided by Promega and Boehringer Mannheim. Chromatographic standards and prepacked Sephadex G-50 columns were from Pharmacia. Reagents and DEAE-cellulose membranes for western blotting were purchased from Sigma, Millipore, and Schleicher & Schuell. Other chemicals, including antibiotics and electrophoretic reagents, were of a molecular biology grade and were obtained from Sigma and Pharmacia.Chloroplast FBPase was obtained from pea leaves according to the method of ; pea Trxs m and f were purified from leaves, as described by Prado et al.

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“…Multiple thioredoxin sequences have also been determined in higher plants, where the proteins are nuclear encoded. Chloroplastic thioredoxins (/ and m type) are synthesized as precursor proteins with a transit sequence of variable size (30-60 amino acids) (Kamo et al, 1989;Lepiniec et al, 1992;Wedel et al, 1992;Lopez-Jaramillo et al, 1994;Stein et al, 1995;Trevanion & Ashton, 1995). The systematic sequencing program of Arabidopsis thaliana cDNA (Hofte et al, 1993, Cooke et al, 1996, Newman et al, 1994 and Oryza sativa (Uchimiya et al, 1993, Umeda et al, 1994 has added numerous new plant sequences.…”

Section: Sequences and Evolutionmentioning

confidence: 99%

Thioredoxins: structure and function in plant cells

1997

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SUMMARY Thioredoxins are ubiquitous small‐molecular‐weight proteins (typically 100–120 amino‐acid residues) containing an extremely reactive disulphide bridge with a highly conserved sequence ‐Cys‐Gly(Ala/Pro)‐Pro‐Cys‐. In bacteria and animal cells, thioredoxins participate in multiple reactions which require reduction of disulphide bonds on selected target proteins/ enzymes. There is now ample biochemical evidence that thioredoxins exert very specific functions in plants, the best documented being the redox regulation of chloroplast enzymes. Another area in which thioredoxins are believed to play a prominent role is in reserve protein mobilization during the process of germination. It has been discovered that thioredoxins constitute a large multigene family in plants with different‐subcellular localizations, a unique feature in living cells so far. Evolutionary studies based on these molecules will be discussed, as well as the available biochemical and genetic evidence related to their functions in plant cells. Eukaryotic photosynthetic plant cells are also unique in that they possess two different reducing systems, one extrachloroplastic dependent on NADPH as an electron donor, and the other one chloroplastic, dependent on photoreduced ferredoxin. This review will examine in detail the latest progresses in the area of thioredoxin structural biology in plants, this protein being an excellent model for this purpose. The structural features of the reducing enzymes ferredoxin thioredoxin reductase and NADPH thioredoxin reductase will also be described. The properties of the target enzymes known so far in plants will be detailed with special emphasis on the structural features which make them redox regulatory. Based on sequence analysis, evidence will be presented that redox regulation of enzymes of the biosynthetic pathways first appeared in cyanobacteria possibly as a way to cope with the oxidants produced by oxygenic photosynthesis. It became more elaborate in the chloroplasts of higher plants where a co‐ordinated functioning of the chloroplastic and extra chloroplastic metabolisms is required.

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Cloning and Sequencing of a Pea cDNA Fragment Coding for Thioredoxin m

Cited by 11 publications

References 2 publications

Interaction of thioredoxins with target proteins: Role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2‐oxoacid dehydrogenase complexes

Interaction of thioredoxins with target proteins: Role of particular structural elements and electrostatic properties of thioredoxins in their interplay with 2‐oxoacid dehydrogenase complexes

High-Yield Expression of Pea Thioredoxin m and Assessment of Its Efficiency in Chloroplast Fructose-1,6-Bisphosphatase Activation

Thioredoxins: structure and function in plant cells

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