The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity.
The sequencing of the genome of Arabidopsis thaliana revealed that this plant contained numerous isoforms of thioredoxin (Trx), a protein involved in thiol-disulfide exchanges. On the basis of sequence comparison, seven putative chloroplastic Trxs have been identified, four belonging to the m-type, two belonging to the f-type, and one belonging to a new x-type. In the present work, these isoforms were produced and purified as recombinant proteins without their putative transit peptides. Their activities were tested with two known chloroplast thioredoxin targets: NADP-malate dehydrogenase and fructose-1,6-bisphosphatase and also with a chloroplastic 2-Cys peroxiredoxin. The study confirms the strict specificity of fructose-bisphosphatase for Trx f, reveals that some Trxs are unable to activate NADP-malate dehydrogenase, and shows that the new x-type is the most efficient substrate for peroxiredoxin while being inactive toward the two other targets. This suggests that this isoform might be specifically involved in resistance against oxidative stress. Three-dimensional modeling shows that one of the m-type Trxs, Trx m3, which has no activity with any of the three targets, exhibits a negatively charged surface surrounding the active site. A green fluorescent protein approach confirms the plastidial localization of these Trxs.
Collin
Valerie
V
Institut de Biotechnologie des Plantes, UMR CNRS 8618, Bât 630, Cedex, France.
Issakidis-Bourguet
Emmanuelle
E
Marchand
Christophe
C
Hirasawa
Masakazu
M
Lancelin
Jean-Marc
JM
Knaff
David B
DB
Miginiac-Maslow
Myroslawa
M
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+); D008958:Models, Molecular; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000378:metabolism; D054464:Peroxiredoxins; D018087:Plastids / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D029681:Arabidopsis Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013879:Thioredoxins; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D010544:Peroxidases; D054464:Peroxiredoxins; D006597:Fructose-Bisphosphatase