Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes.
A new over-expression system has been set up for Escherichia coli thioredoxin, yielding 55 mg purified protein/10 g fresh cells. This system has been used to produce thioredoxin modified by site-directed mutagenesis. Taking advantage of the structural and enzymatic similarity between E. coli and spinach m-type thioredoxin, Asp61 of E. coli thioredoxin has been changed into Asn in order to investigate the impact of the suppression of a charged residue on the interaction of thioredoxin with target enzymes. The modification did not significantly alter the structure of the protein. Neither the rate of reduction of insulin and 5,5'-dithio-bis(2-nitrobenzoic acid) by the reduced thioredoxin, nor the reduction by NADPH-dependent thioredoxin reductase, have been modified. The major effect of the mutation was observed for chloroplast enzyme activation with thioredoxin reduced by dithiothreitol and with thioredoxin reduced by ferredoxin-dependent thioredoxin reductase in a light-activation reconstituted chloroplast system. The substitution of the negatively charged Asp61 by the neutral Asn led to an increase in the efficiency of spinach fructose-1,6-bisphosphatase activation by the dithiothreitol-reduced thioredoxin, and to an increase in both spinach fructose-1,6-bisphosphatase and corn NADP-dependent malate dehydrogenase activities in the light-activation system. This suggests that the suppression of the negative charge improves the reactivity of thioredoxin with chloroplast enzymes such as fructose-1,6-bisphosphatase and ferredoxin-dependent thioredoxin reductase.
de Lamotte-Guery
F
F
Laboratoire de Physiologie Végétale Moléculaire, Université Paris Sud, Orsay, France.
Miginiac-Maslow
M
M
Decottignies
P
P
Stein
M
M
Minard
P
P
Jacquot
J P
JP
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics*; D001483:Base Sequence; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010944:Plants / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism
D054479:Chloroplast Thioredoxins; D010940:Plant Proteins; D001224:Aspartic Acid; D013879:Thioredoxins; D001216:Asparagine; D008291:Malate Dehydrogenase; D006597:Fructose-Bisphosphatase