Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties.
Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37 degrees C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309 + or - 35 nkat mg(-1)) and GS1b (1,166 + or - 65 nkat mg(-1)) enzymes were obtained by decreasing the expression temperature to 10 degrees C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.
de la Torre
Fernando
F
Departamento de Biología Molecular y Bioquímica, Instituto Andaluz de Biotecnología, Unidad Asociada UMA-CSIC, Universidad de Málaga, Campus de Teatinos, E-29071 Málaga, Spain.
García-Gutiérrez
Angel
A
Crespillo
Remedios
R
Cantón
Francisco R
FR
Avila
Concepción
C
Cánovas
Francisco M
FM
D001419:Bacteria / Q000235:genetics*; D002384:Catalysis; D003600:Cytosol / Q000378:metabolism; D004795:Enzyme Stability; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D028223:Pinus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D020035:Holoenzymes; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005974:Glutamate-Ammonia Ligase