Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme.

摘要

cDNAs corresponding to the 2 cytosolic glutamine synthetase (GS) [glutamate-ammonia ligase] polypeptides (a and b) of Medicago truncatula were cloned and sequenced. Using these 2 cDNAs, a construct was prepared encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which would assemble to give an enzyme containing chimaeric active sites. The native and the domain-swapped enzymes were expressed in E. coli, where they were catalytically andphysiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. Slight differences occurred inthe kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able toself-assemble, perhaps randomly, to form heteromeric isoenzymes. Studies on the GS isoenzymes of nodules, roots, stems and stipules showed that heteromeric isoenzymes occurred in the plant. Nucleotide sequence data have been submitted to the EMBL/GenBank/DDBJ databases under the accession numbers Y10267 (MtGSa cDNA) and Y10268 (MtGSb cDNA).