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

Two cDNA clones for glutamine synthetase (GS), Clgln1 and Clgln2, were isol ated from a Canavalia lineata cDNA library constructed with poly(A)+ RNA fr om the mature plant leaves. From a comparison of the primary structures, Cl gln1 was judged to encode a cytosolic isoform and Clgln2 to encode a plasti dic isoform. It was revealed by those sequence comparisons that the two cys teine residues (Cys-306 and Cys-371) of Clgln2 (chloroplastic GS encoding g ene) were substituted by alanine and serine in the Clgln1 clone (cytosolic GS encoding gene), respectively. These cysteine substitutions were found be tween chloroplastic GS (GS2) and cytosolic GS (GS1) sequences of all plants . These unique cysteine residues may account for the specific susceptibilit y of the plastidic GS by sulfhydryl reagents. To investigate this assumptio n the two additional cysteine residues of GS2 were mutated combinatorially and the resulting recombinant GS proteins as well as the two wild-type cyto solic GS and chloroplastic GS were examined in vitro, only GS2, of the wild -type forms, was significantly activated by dithiotreitol. Moreover, the mu tant 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 th e two were also activated. (C) 1999 Elsevier Science Ireland Ltd. All right s reserved.