The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

Abstract

The three-dimensional structure of the hexameric (␣␤) 6 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex ironsulfur flavoprotein. In the hexameric species, interprotomeric ␣-␣ and ␣-␤ contacts are mediated by the C-terminal domain of the ␣ subunit, which is based on a ␤ helical fold so far unique to glutamate synthases. The ␣␤ protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the ␤ subunit, to the FMN on the ␣ subunit, through the low potential [4Fe-4S] 1؉/2؉ centers on the ␤ subunit and the [3Fe-4S] 0/1؉ cluster on the ␣ subunit. The (␣␤) 6 hexamer exhibits a concentration-dependent equilibrium with ␣␤ monomers and (␣␤) 2 dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP ؉ . Hexamerization seems to decrease the catalytic efficiency of the ␣␤ protomer only 3-fold by increasing the K m values measured for L-Gln and 2-OG. However, it cannot be ruled out that the (␣␤) 6 hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.Glutamate synthases (GltS) 2 are complex iron-sulfur flavoproteins that catalyze the reductive transfer of the L-Gln amide group to the C2 carbon of 2-oxoglutarate (2-OG), yielding two molecules of L-glutamate (L-Glu). GltS are found in bacteria, yeast, and plants, where they form with glutamine synthetase an essential pathway for ammonia assimilation (1-3). Bacterial NADPH-dependent GltS (NADPH-GltS) is formed by one ␣ subunit (␣GltS) and one ␤ subunit (␤GltS) of 162 and 52 kDa, respectively, for the Azospirillum brasilense GltS. The ␣␤ protomer contains one FAD (on ␤GltS), one FMN (on ␣GltS), and three different iron-sulfur clusters (one [3Fe-4S] 0/1ϩ cluster on ␣GltS and two [4Fe-4S] 1ϩ/2ϩ centers on ␤GltS; Fig. 1A). On the basis of sequence, structural, and mechanistic similarities, the NADPH-GltS serves as a model for the other two main forms of GltS, namely (i) the ferredoxin-dependent GltS found in cyanobacteria and photosynthetic tissues of plants, which is similar to ␣GltS and (ii) the eukaryotic type of GltS, which is NADH-dependent and is found in yeast, nonphotosynthetic tissues of plants, and lower eukaryotes; this GltS species is formed by a single polypeptide chain derived from the fusion of bacterial ␣ and ␤ subunits.Several lines of evidence support an essential role of GltS in all of these cell types, making it a potential target of novel drugs. For example, the NADPH-GltS has been found to be essential in Mycobacterium tuberculosis, where it has been shown that Ͻ1 M azaserine, a GltS inhibitor, is sufficient t...

Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure

“…The complex contains two FAD, one [2Fe-2S] cluster, and two [4Fe-4S] clusters. NfnA shows sequence similarities to plant ferredoxin: NADP ϩ reductase (Fnr) and bacterial dihydroorotate dehydrogenase (Dodh) from bacteria and NfnB to the ␤-subunit of bacterial NADPH-dependent glutamate synthase (Gls) (22) and eukaryotic dihydropyrimidine dehydrogenase (Dpdh) (23). The transhydrogenase complex catalyzes the reversible reduction of ferredoxin and NAD ϩ with 2 NADPH (Reaction 2).…”

“…Coot was used for electron density analysis and model building (37). Several secondary structure elements were clearly visible in the experimental electron density, and the related subunits of Dodh from Lactococcus lactis (38) and Gls from Azospirillum brasilense (22) were fitted into the electron density of NfnA and NfnB, respectively. Automatic model building in Phenix (39) correctly positioned ϳ50% of the residues of NfnA and NfnB into the electron density when incorporating the superimposed Dodh and Gls as reference models.…”

Comparison of single‐particle analysis and electron tomography approaches: an overview

“…(B–D) Use of single‐particle analysis to determine the 3D structural architecture of the multi‐enzymatic complex Glutamate synthase (GltS) from E. coli [the figure was produced with the data used in Sorzano et al . (2007a); the GltS function was interpreted in Cottevieille et al (2008) based on these data]. (B) 2D cryo‐EM projection image of the GltS sample on a holey carbon film vitrified by plunge freezing in liquid ethane.…”

“…The GltS are found in bacteria, yeast and plants, where they form with glutamine synthetase an essential pathway for ammonia assimilation. The technique used to compute the 3D electron density map of the whole complex is described in Sorzano et al (2007a) and the interactions and function of the GltS sub‐units are discussed in Cottevieille et al (2008). The structure was computed from low‐dose cryo‐EM images of a GltS sample on a holey carbon film vitrified by plunge freezing in liquid ethane (Fig.…”

Elastic image registration to fully explore macromolecular dynamics by electron microscopy

“…Complex image processing algorithms are required to compute the structure from the images. When the angular distribution of singleparticle orientations samples Fourier space completely and the population is homogeneous, standard image processing strategies allow computing an average structure at resolution of 0.4-1 nm [3,4].…”