Identification of the cysteine residues involved in redox modification of plant plastidic glucose-6-phosphate dehydrogenase.
The cDNA sequences encoding cytosolic and light-modulated plastidic glucose-6-phosphate dehydrogenase (G6PDH) from potato were modified by polymerase chain reaction and subsequently overexpressed in Escherichia coli. Characterization of the recombinant enzymes showed that they closely resembled their native counterparts. Treatment with reduced dithiothreitol or glutathione led to inactivation of plastidic G6PDH, whereas the activity of the cytosolic isoenzyme was not influenced by reduction. As for the native enzyme, inactivation of recombinant plastidic G6PDH was accelerated by thioredoxin m and could be fully reversed by subsequent addition of oxidant. To identify the residues which are involved in redox regulation of plastidic G6PDH, each of the six cysteines in the mature protein sequence was exchanged separately for serine by site-directed mutagenesis. Two mutant proteins exhibited characteristics of the reduced wild-type enzyme. Exchange of either Cys149 or Cys157 to serine abolished the regulatory properties, suggesting that these cysteine residues are the sites responsible for redox-mediated inactivation of plastidic G6PDH.
Wenderoth
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Pflanzenphysiologie, FB 5 Biologie/Chemie, Universität Osnabrück, D-49069 Osnabrück, Germany.
Scheibe
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R
von Schaewen
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A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology; D003545:Cysteine; D017931:DNA Primers; D004926:Escherichia coli; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018087:Plastids / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology
D017931:DNA Primers; D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase; D003545:Cysteine