Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes

Abstract

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. Copyright © 1991, Wiley Blackwell. All rights reserved