The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin.
The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.
Institut de Biotechnologie des Plantes, UMR 8618 CNRS, Université de Paris-Sud, Orsay, France.
A
A
Goyer
P
P
Decottignies
S
S
Lemaire
E
E
Ruelland
E
E
Issakidis-Bourguet
J P
JP
Jacquot
M
M
Miginiac-Maslow
D000818:Animals; D002696:Chlamydomonas / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D016297:Mutagenesis, Site-Directed / Q000235:genetics; D018515:Plant Leaves / Q000201:enzymology*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D010940:Plant Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D004228:Dithionitrobenzoic Acid; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D003545:Cysteine