IDENTIFICATION AND CHARACTERIZATION OF THE 2ND REGULATORY DISULFIDE BRIDGE OF RECOMBINANT SORGHUM LEAF NADP-MALATE DEHYDROGENASE

Unique among malate dehydrogenases, the NADP-dependent chloroplastic f orm undergoes a reductive activation in the light. This process is thi oredoxin-mediated and involves at least two disulfides. Only one of th em, situated near the N terminus, has been localized. The enzyme also bears 2 cysteines at the C terminus. The possible role of these cystei nes was investigated by replacing them separately, or together, by ala nines, by site-directed mutagenesis. The proteins altered at the C ter minus were still dithiol-dependent for full activation, with activatio n kinetics similar to those of the wild type enzyme. However, they exh ibited a weak activity in the oxidized form with a dramatically increa sed K(m) for oxalacetate. Their activation was not inhibited by NADP. When C-terminal Cys mutations were combined with N-terminal Cys mutati ons, 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 th ioredoxin-dependent regulatory disulfide of NADP-malate dehydrogenase. Integrating our data about the characteristics of each of the regulat ory disulfides and information from three-dimensional structure modeli ng, we propose a model for the redox control of NADP-malate dehydrogen ase.