Crystallization and preliminary crystallographic characterization of glutamine synthetase from Medicago truncatula

Abstract

The condensation of ammonium and glutamate into glutamine catalyzed by glutamine synthetase (GS) is a fundamental step in nitrogen metabolism in all kingdoms of life. In plants, this is preceded by the reduction of inorganic nitrogen to an ammonium ion and therefore effectively articulates nitrogen fixation and metabolism. Although the three-dimensional structure of the dodecameric bacterial GS was determined quite some time ago, the quaternary architecture of the plant enzyme has long been assumed to be octameric, mostly on the basis of low-resolution electron-microscopy studies. Recently, the crystallographic structure of a monocotyledonous plant GS was reported that revealed a homodecameric organization. In order to unambiguously establish the quaternary architecture of GS from dicotyledonous plants, GS1a from the model legume Medicago truncatula was overexpressed, purified and crystallized. The collection of synchrotron diffraction data to 2.35 A resolution allowed the determination of the three-dimensional structure of this enzyme by molecular replacement.

Figure 1

Coomassie Blue-stained 12.5% SDS–PAGE of the purified recombinant GS1a from M. truncatula used for crystallization trials. Lane 1, molecular-mass markers. The apparent molecular mass (in kDa) of the marker proteins is given on the left. Lanes 2–5 contained 1, 2.5, 5 and 10 µg of recombinant GS1a, respectively.

Figure 2

Single crystals of native GS1a from M. truncatula belonging to the monoclinic space group P2₁.