Cysteine-153 is required for redox regulation of pea chloroplast fructose-1,6-bisphosphatase.
Chloroplastic fructose-1,6-bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys49, Cys153, Cys173, Cys178 and Cys190) have been modified individually into serine residues by site-directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild-type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin h system). Finally, the C49S mutant is essentially identical to the wild-type enzyme. These results are discussed in the light of recent crystallographic data obtained on spinach FBPase [Villeret et al. (1995) Biochemistry 34, 4299-4306].
Jacquot
J P
JP
Institut de Biotechnologie des Plantes, URA 1128 CNRS, Université de Paris-Sud, Orsay, France. jacquot@ibp.u-psud.fr
Lopez-Jaramillo
J
J
Miginiac-Maslow
M
M
Lemaire
S
S
Cherfils
J
J
Chueca
A
A
Lopez-Gorge
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D003545:Cysteine / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D006597:Fructose-Bisphosphatase / Q000235:genetics / Q000378:metabolism*; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000494:pharmacology
D054479:Chloroplast Thioredoxins; D011994:Recombinant Proteins; D013879:Thioredoxins; D006597:Fructose-Bisphosphatase; D003545:Cysteine; D004229:Dithiothreitol