Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme.
We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. 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 produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically 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. We have found slight differences in the 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 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.
Carvalho
H
H
Centro de Citologia Experimental da Universidade do Porto, Portugal.
Sunkel
C
C
Salema
R
R
Cullimore
J V
JV
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D000455:Medicago sativa / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D007527:Isoenzymes; D010455:Peptides; D011993:Recombinant Fusion Proteins; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase