Identification and characterization of the second regulatory disulfide bridge of recombinant sorghum leaf NADP-malate dehydrogenase.

Abstract Unique among malate dehydrogenases, the NADP-dependent chloroplastic form undergoes a reductive activation in the light. This process is thioredoxin-mediated and involves at least two disulfides. Only one of them, situated near the N terminus, has been localized. The enzyme also bears 2 cysteines at the C terminus. The possible role of these cysteines was investigated by replacing them separately, or together, by alanines, by site-directed mutagenesis. The proteins altered at the C terminus were still dithiol-dependent for full activation, with activation kinetics similar to those of the wild type enzyme. However, they exhibited a weak activity in the oxidized form with a dramatically increased Km for oxalacetate. Their activation was not inhibited by NADP. When C-terminal Cys mutations were combined with N-terminal Cys mutations, permanently active, thioredoxin-independent enzymes were obtained. They exhibited the biochemical properties of the activated wild type protein. Clearly, the 2 C-terminal cysteines constitute the second thioredoxin-dependent regulatory disulfide of NADP-malate dehydrogenase. Integrating our data about the characteristics of each of the regulatory disulfides and information from three-dimensional structure modeling, we propose a model for the redox control of NADP-malate dehydrogenase.