Sites of interaction of thioredoxin with sorghum NADP-malate dehydrogenase.
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.
Goyer
A
A
Institut de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris-Sud, Orsay, France.
Decottignies
P
P
Issakidis-Bourguet
E
E
Miginiac-Maslow
M
M
D001665:Binding Sites; D015153:Blotting, Western; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D016296:Mutagenesis; D006109:Poaceae / Q000201:enzymology*; D013879:Thioredoxins / Q000378:metabolism*
D004220:Disulfides; D013879:Thioredoxins; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)