HETEROMERIC ASSEMBLY OF THE CYTOSOLIC GLUTAMINE-SYNTHETASE POLYPEPTIDES OF MEDICAGO-TRUNCATULA - COMPLEMENTATION OF A GLNA ESCHERICHIA-COLIMUTANT WITH A PLANT DOMAIN-SWAPPED ENZYME

We have cloned and sequenced the cDNAs corresponding to the two cytoso lic glutamine synthetase (GS) polypeptides (a and b) of Medicago trunc atula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to pro duce a domain-swapped polypeptide which should assemble to give an enz yme containing chimeric active sites. Both the native and the domain-s wapped enzymes were expressed in Escherichia coli where they were cata lytically and physiologically active as they were able to rescue a gln A deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on io n-exchange chromatography. We have found slight differences in the kin etic properties of the purified enzymes and in the modulation of their activities by several putative cellular effecters. In vitro dissociat ion of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.