The internal Cys‐207 of sorghum leaf NADP‐malate dehydrogenase can form mixed disulphides with thioredoxin

FEBS Letters

Decottignies, Paulette¹  Ruelland, Eric¹  Miginiac-Maslow, Myroslawa¹  Lemaire, Stephane¹  Goyer, Aymeric¹  Issakidis-Bourguet, Emmanuelle¹  Jacquot, Jean-Pierre¹ 

[1] Institut de Biotechnologie des Plantes, UMR 8618 CNRS, Bâtiment 630, Université de Paris-Sud, 91405 Orsay Cedex, France

关键词: Disulfide;    NADP-malate dehydrogenase;    Site-directed mutagenesis;    Thiol;    Thioredoxin;    DTNB;    5;    5′-dithiobis(2-nitrobenzoic acid);    TNB;    5-thio(2-nitrobenzoic acid);    DTT;    dithiothreitol;    NADP-MDH;    NADP-dependent malate dehydrogenase;    Trx;    thioredoxin;    Δ-15 MDH;    MDH with the 15 most N-terminal residues deleted;    ΔN MDH;    MDH with the 33 most N-terminal residues deleted;    NDM MDH;    MDH mutated at the two most N-terminal cysteines 24 and 29;   

DOI  :  10.1016/S0014-5793(99)00051-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown