The plastidic glutamine synthetase activity is directly modulated by means of redox change at two unique cysteine residues

Two cDNA clones for glutamine synthetase (GS), Clgln1 and Clgln2, were isolated from a Canavalia lineata cDNA library constructed with poly(A)+ RNA from the mature plant leaves. From a comparison of the primary structures, Clgln1 was judged to encode a cytosolic isoform and Clgln2 to encode a plastidic isoform. It was revealed by those sequence comparisons that the two cysteine residues (Cys-306 and Cys-371) of Clgln2 (chloroplastic GS encoding gene) were substituted by alanine and serine in the Clgln1 clone (cytosolic GS encoding gene), respectively. These cysteine substitutions were found between chloroplastic GS (GS2) and cytosolic GS (GS1) sequences of all plants. These unique cysteine residues may account for the specific susceptibility of the plastidic GS by sulfhydryl reagents. To investigate this assumption the two additional cysteine residues of GS2 were mutated combinatorially and the resulting recombinant GS proteins as well as the two wild-type cytosolic GS and chloroplastic GS were examined in vitro, only GS2, of the wild-type forms, was significantly activated by dithiotreitol. Moreover, the mutant form, mutated at both of the two additional cysteine residues, was not activated by the reductant, but the mutant forms mutated at only one of the two were also activated. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.