Light-modulated NADP-malate dehydrogenases from mossfern and green algae: insights into evolution of the enzyme's regulation

Chloroplast NADP-dependent malate dehydrogenase is one of the best-studied light-regulated enzymes. In C3 plants, NADP-MDH is a part of the 'malate va lve' that controls the export of reducing equivalents in the form of malate to the cytosol. NADP-MDH is completely inactive in the dark and is activat ed in the light with reduced thioredoxin. Compared with its permanently act ive NAD-limited counterparts, NADP-MDH exhibits N- and C-terminal sequence extensions, each bearing one regulatory disulphide. Upon reduction of the C -terminal disulphide, the enzyme active site becomes accessible for the sub strate. Reduction of the N-terminal disulphide promotes a conformational ch ange advantageous for catalysis. To trace the evolutionary development of t his intricate regulation mechanism, we isolated cDNA clones for NADP-MDH fr om the mossfern Selaginella and from two unicellular green algae. While the NADP-MDH sequence from Selaginella demonstrates the classic cysteine patte rn of the higher plant enzyme, the sequences from the green algae are devoi d of the N-terminal regulatory disulphide. Phylogenetic analysis of new seq uences and of those available in the databases led to the conclusion that t he chloroplast NADP-MDH and the cytosolic NAD-dependent form arose via dupl ication of an ancestral eubacterial gene, which preceded the separation of plant and animal lineages. Redox-sensitive NADP-MDH activity was detected o nly in the 'green' plant lineage starting from the primitive prasinophytic algae but not in cyanobacteria, Cyanophora paradoxa, red algae and diatoms. The latter organisms therefore appear to utilize mechanisms other than the light-regulated 'malate valve' to remove from plastids excessive electrons produced by photosynthesis. (C) 2000 Elsevier Science B.V. All rights rese rved.