Identification and characterization of the second regulatory disulfide bridge of recombinant sorghum leaf NADP-malate dehydrogenase.
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.
Issakidis
E
E
Laboratoire de Physiologie Végétale Moléculaire, Unité de Recherche associée 1128 Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.
Saarinen
M
M
Decottignies
P
P
Jacquot
J P
JP
Crétin
C
C
Gadal
P
P
Miginiac-Maslow
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology; D003196:Computer Graphics; D003545:Cysteine; D004220:Disulfides / Q000032:analysis*; D004789:Enzyme Activation; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism* / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D006109:Poaceae / Q000201:enzymology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D009249:NADP; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D003545:Cysteine