Thioredoxin-h1 reduces and reactivates the oxidized cytosolic malate dehydrogenase dimer in higher plants.
Cytosolic malate dehydrogenase (cytMDH) was captured by thioredoxin affinity chromatography as a possible target protein of cytosolic thioredoxin (Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004) Plant Cell Physiol. 45, 18-27). To further dissect this interaction, we aimed to determine whether cytMDH can interact with the cytosolic thioredoxin and whether its activity is redox-regulated. We obtained the active recombinant cytMDH that could be oxidized and rendered inactive. Inactivation was reversed by incubation with low concentrations of dithiothreitol in the presence of recombinant Arabidopsis thaliana thioredoxin-h1. Inactivation of cytMDH was found to result from formation of a homodimer. By cysteine mutant analysis and peptide mapping analysis, we were able to determine that the cytMDH homodimer occurs by formation of a disulfide bond via the Cys(330) residue. Moreover, we found this bond to be efficiently reduced by the reduced form of thioredoxin-h1. These results demonstrate that the oxidized form cytMDH dimer is a preferable target protein of the reduced form thioredoxin-h1 as suggested by thioredoxin affinity chromatography.
Hara
Satoshi
S
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama 226-8503, Japan.
Motohashi
Ken
K
Arisaka
Fumio
F
Romano
Patrick G N
PG
Hosoya-Matsuda
Naomi
N
Kikuchi
Nobuhiro
N
Fusada
Naoki
N
Hisabori
Toru
T
D017360:Arabidopsis / Q000201:enzymology*; D002846:Chromatography, Affinity; D003545:Cysteine / Q000032:analysis / Q000235:genetics; D003600:Cytosol / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D008291:Malate Dehydrogenase; D003545:Cysteine; D004229:Dithiothreitol