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

• Published in: The Journal of biological chemistry 1994-02, Vol.269 (5), p.3511-3517
• Main Authors: Issakidis, E, Saarinen, M, Decottignies, P, Jacquot, J.P, Cretin, C, Gadal, P, Miginiac-Maslow, M
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; CHLOROPLASTE; Chloroplasts - enzymology; CISTEINA; CLOROPLASTO; Computer Graphics; CYSTEINE; Disulfides - analysis; Enzyme Activation; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Malate Dehydrogenase (NADP+); Malate Dehydrogenase - chemistry; Malate Dehydrogenase - metabolism; MALATE DESHYDROGENASE; MALATO DESHIDROGENASA; Models, Molecular; Molecular Sequence Data; MUTACION; MUTACION INDUCIDA; Mutagenesis, Site-Directed; MUTANT; MUTANTES; MUTATION; MUTATION PROVOQUEE; NADP - metabolism; NADP - pharmacology; Oligodeoxyribonucleotides; OXIDACION; Oxidoreductases; OXYDATION; Poaceae - enzymology; Protein Structure, Secondary; REACCIONES QUIMICAS; REACTION CHIMIQUE; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; REDUCCION; REDUCTION; SORGHUM BICOLOR
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)41892-8

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