An internal cysteine is involved in the thioredoxin-dependent activation of sorghum leaf NADP-malate dehydrogenase.
The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins. Previous experiments showed that four cysteines located in specific N- and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified. The C29S/C365A/C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys24-Cys207 disulfide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.
Ruelland
E
E
Institut de Biotechnologie des Plantes, ERS 569 CNRS, Bâtiment 630, Université de Paris-Sud, 91405 Orsay Cedex, France.
Lemaire-Chamley
M
M
Le Maréchal
P
P
Issakidis-Bourguet
E
E
Djukic
N
N
Miginiac-Maslow
M
M
D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D008027:Light; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008958:Models, Molecular; D016296:Mutagenesis; D013879:Thioredoxins / Q000378:metabolism*
D004220:Disulfides; D013879:Thioredoxins; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D003545:Cysteine