Abstract

After the discovery of the light-activation properties of the chloroplastic NADP-dependent malate dehydrogenase in 1970, the elements of the activation pathway were identified and shown to consist of the stromal proteins of the ferredoxin/thioredoxin system. It was further demonstrated that the activation was a reductive process during which disulfides were reduced into dithiols by reduced thioredoxin. Sequence alignments with the permanently active NAD-malate dehydrogenases revealed N- and C-terminal extensions specific for the light-regulated form. A regulatory disulfide was identified in the amino-terminal extension by chemical derivatisation: its reduction was correlated to the activation of the enzyme. The use of site-directed mutagenesis techniques revealed the complexity of the intramolecular activation mechanism, showing that two different disulfides were reduced per subunit of this homodimeric enzyme: one located in the N-terminal extension, the other in the C-terminal extension. A model was proposed where the C-terminal extension locks the access to the active site, whereas the N-terminal extension governs the conformation of the active site. The identification of the catalytic histidine allowed us to test the accessibility of the active site and to demonstrate the validity of the proposed model.