Light-dependent activation of NADP-malate dehydrogenase: a complex process

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 Nand 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 tc:st the accessibility of the active site and to demonstrate the validity of the proposed model.