Journal Article

Functional Expression of Two Pine Glutamine Synthetase Genes in Bacteria Reveals that they Encode Cytosolic Holoenzymes with Different Molecular and Catalytic Properties

Fernando de la Torre, Angel García-Gutiérrez, Remedios Crespillo, Francisco R. Cantón, Concepción Ávila and Francisco M. Cánovas

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 7, pages 802-809
Published in print July 2002 | ISSN: 0032-0781
Published online July 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf094
Functional Expression of Two Pine Glutamine Synthetase Genes in Bacteria Reveals that they Encode Cytosolic Holoenzymes with Different Molecular and Catalytic Properties

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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°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±35 nkat mg–1) and GS1b (1,166±65 nkat mg–1) enzymes were obtained by decreasing the expression temperature to 10°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.

Keywords: Keywords: Conifers — Pinus — Nitrogen metabolism — Recombinant isoenzymes — Negative cooperativity.

Journal Article.  5059 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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