Mariana Stein

Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins, purification and biochemical properties

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 11817 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.

Residue Glu-91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin-thioredoxin reductase

The [2Fe‐2S] soluble ferredoxin from Chlamydomonas reinhardtii was mutated by site directed mutagenesis, using PCR and the expression plasmid pET‐Fd as a template. The recombinant mutated proteins were purified to homogeneity and tested in the activation of NADP‐malate dehydrogenase, a light dependent reaction in which ferredoxin thioredoxin reductase (FTR) and thioredoxin are involved. The mutation of residue Glu‐91 (E92 in spinach, E94 in Anabaena) alone, either to Gln (E91Q) or to Lys (E91K), was found to completely abolish the reaction of the enzyme light activation. On the other hand, the mutants (E92Q) or (E92K) were as efficient as the wild type ferredoxin in this reaction whereas the double mutants (E91Q/E92Q) or (E91K/E92K) had no activity. In addition, a triple mutant (D25A/E28Q/E29Q) was also found to be inactive for this redox dependent light activation. All these mutations had much weaker effects on the ferredoxin/ferredoxin NADP reductase interaction as measured by the cytochrome c reduction assay. These results indicate that there is a recognition site for FTR in the C terminus part of ferredoxin, but also that a core of negatively charged residues in the α1 helix of ferredoxin might be important in the general process of light activation.

NMR structures of ferredoxin chloroplastic transit peptide from Chlamydomonas reinhardtii promoted by trifluoroethanol in aqueous solution

The 32‐amino acid transit peptide of the unicellular green alga Chlamydomonas reinhardtii ferredoxin has been synthesized and analysed by NMR spectroscopy and circular dichroism. The results show that while the peptide is unstructured in water, it undergoes an α‐helix formation from residue 3 to 13 in a 30:70 molar‐ratio mixture of 2,2,2‐trifluoroethanol. The remainder of the peptide is still unstructured in CF3CD2OD/H2O mixtures, but is distributed on a side opposite to a hydrophobic ridge formed by Met5, Phe9 and Val13 on the induced a‐helix. The NMR structures driven by 2,2,2‐trifluoroethanol in aqueous solution, are discussed in terms of potent interactions with the chloroplast envelope and its translocation molecular machinery.

A cDNA clone encoding Chlamydomonas reinhardtii preferredoxin

Fds are ubiquitous low mo1 wt proteins containing iron sulfur center(s) involved in numerous electron transfer reactions. In plants, one of the major functions of Fd is to provide the reducing power for chloroplastic NADP photoreduction. In addition, Fd is also needed for nitrite, ammonia, and sulfite assimilation as well as for pseudocyclic electron flow and fatty acid metabolism and for the light regulation of chloroplastic enzymes (Orme-Johnson, 1973). This protein is encoded by nuclear genes and is, therefore, produced as a precursor that is subsequently cleaved inside the chloroplast (Smeekens et al., 1985). We have previously purified Chlamydomonas reinhardtii Fd and determined its primary structure by direct amino acid sequencing (Schmitter et al., 1988). Based on this sequence and using the polymerase chain reaction, we have isolated a nucleotidic sequence coding for the mature portion of C. reinhardtii Fd and shown that the deduced amino acid sequence was identical with only one substitution Thr7 to Ser (Table I). In addition, we demonstrated that Escherichia coli cells were able to direct the synthesis of C. reinhardtii Fd polypeptide and to reassemble it together with an iron-sulfur center (Rogers et al., 1992). In this paper we report the sequence of a cDNA encoding C. reinhardtii preferredoxin. A Xgtll cDNA was sequenced (527 bp) and found to encode a 126-amino acid precursor with a molecular mass of 13,250 D, compared to the mature form, which contains 94 amino acids and has a molecular mass of 9,908 D. The deduced amino acid sequence was completely homologous to the sequence reported by Schmitter et al. (1988). In addition, the sequence revealed the structure of the 32-amino acid transit peptide (molecular mass 3342 D), which is as follows: MAMAMRSTFAARVGAKPAVRGARPASRMSCMA. Severa1 lines of evidence indicate that the first ATG is the initiation codon. First, it is preceded by TCA and AAA, triplets theoretically coding for Ser and Lys but rarely if ever used for nuclear genes in C. reinhardtii. Second, the flanking sequence surrounding the initiation codon (AAAAATGGC) fits well the eukaryotic translation initiation consensus (Wedel et al., 1992). Finally, the beginning of the transit peptide (MAMAM) is highly similar to the MAQM sequence reported by Wedel et al. (1992) and to the MAMAT of Hoffmann et al. (1988), and the dipeptide MA is overwhelm-

Molecular Aspects of Components of the Ferredoxin/Thioredoxin Systems

In the chloroplasts of eukaryotic photosynthetic cells, ferredoxin and thioredoxin are linked in a redox cascade which activates several catalysts of the Calvin cycle by molecular reduction of selected disulfide bridges. These two redox constituents are also present in Chlamydomonas reinhardtii and their properties are described. Ferredoxin is a nucleus-encoded protein synthesized as a 126 amino-acid precursor which is processed to a 94 amino-acid mature protein. In contrast with the intronless genes of ferredoxin in land plants, the C. reinhardtii ferredoxin gene (Frx1) contains a single intron and is present in very few copies. In chloroplasts, ferredoxin is located at a metabolic branchpoint. Ferredoxin distributes electrons to many biochemical pathways involving reductive chemistry; e.g. reduced ferredoxin is a substrate of the enzymes ferredoxin NADP+ oxidoreductase (FNR) and ferredoxin-thioredoxin reductase (FTR). Several characteristics of electron transfer from ferredoxin to FNR and FTR will be described. Evidence will also be presented that FNR is subject to post-translational modification (methylation) in C. reinhardtii. As in land plants, C. reinhardtii cells contain several types of thioredoxin, with different subcellular localization. Thioredoxin h is nucleus-encoded, located in the cytosol and does not require a transit sequence. On the other hand, thioredoxin m (106 amino-acids in its mature form) is also nucleus-encoded, synthesized as a precursor of 140 amino-acids and located in the chloroplast. The genes coding for the two thioredoxins (Trx1 and Trx2) have been isolated and sequenced. In addition, the molecular 3D-structures of these two proteins will be presented as well as some of their biochemical characteristics.

Residue GLU-91 of chlamydomonas reinhardtii ferredoxin is essential for the reaction of ferredoxin-nitrite reductase and ferredoxin-glutamate synthase

Chloroplast ferredoxins (Fd) are small monomeric proteins of approximately 10 kDa containing a [2Fe-2S] iron sulfur centre with low redox potential. Ferredoxin is a central protein for transferring electrons from the photosynthetic chain to several enzymes in the chloroplast including nitrite reduction, glutamate formation, sulfite reduction, lipid biosynthesis, chloroplastic enzyme light regulation and also NADP+ photoreduction involved in the CO2 fixation cycle (Amon, 1988). There is a high amino acide sequence similarity generally about 70% among various soluble ferredoxins from cyanobacteria to higher plants (Rypniewski et al., 1991). Ferredoxin forms a strong protein-protein complexe with Fd-dependent enzymes. The most extensively studied systems are ferredoxin-NADP+ oxidoreductase (FNR) (EC 1.18.1.3) and ferredoxin-thioredoxin reductase (FTR). Several ferredoxin residues are supposed to constitute binding site: D26, E29, E30 and D65 for FNR (de Pascalis et al., 1993a) and D34, D65, E92, E93, E94 and A97 for FTR (de Pascalis et al., 1993b).