Abstract
The three-dimensional structure of the hexameric (alphabeta)(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 iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alphabeta 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 beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The (alphabeta)(6) hexamer exhibits a concentration-dependent equilibrium with alphabeta monomers and (alphabeta)(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 alphabeta 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 (alphabeta)(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.
Highlights
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 [1,2,3]
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
Summary
GltS, glutamate synthase; ␣GltS, ␣ subunit of glutamate synthase; GltS,  subunit of glutamate synthase; 2-OG, 2-oxoglutarate; 2-IG, 2-iminoglutarate; EM, electron microscopy; DPD, dihydropyrimidine dehydrogenase; DTT, 1,4-dithiothreitol; Em, midpoint potential; GltSHis, glutamate synthase formed by the wild-type ␣ subunit and a  subunit form carrying a C-terminal hexahistidinyl extension; GltSHis⌬7 or GltSHis⌬40, GltSHis variant resulting from deletion of the C-terminal 7 or 40 residues, respectively, of the ␣ subunit; MetS, L-methionine sulfone; MM, molecular mass; NAD(P)H-GltS, NAD(P)H-dependent glutamate synthase; SAXS, small angle x-ray scattering. A combination of structural and functional experiments has shown that at least five steps at three different locations are necessary to carry out the overall reaction [1,2,3] (Fig. 1A), namely NADPH oxidation (step 1) and transfer of reducing equivalents from FAD (on GltS) to FMN (on ␣GltS) through at least two of the three [Fe-S] clusters (step 2) and binding and hydrolysis of L-Gln at the glutaminase site in the Type II (PurF) glutamine amidotransferase domain of ␣GltS (step 3) followed by transfer of the released ammonia molecule to the synthase site through the intramolecular tunnel (step 4). The loss of coupling of the glutaminase and synthase catalytic subsites within the isolated ␣GltS suggests that a conformational change occurs in this subunit upon association with GltS [16] For all of these reasons, obtaining information on the threedimensional structure of the GltS ␣ protomer is a key step toward the understanding of the sophisticated mechanism of action and regulation of this essential enzyme. Modeling of the (␣) oligomer of NADPH-GltS into the 9.5 Å resolution cryo-EM-derived electron density shows how the C-terminal  helical domain of ␣GltS acts as a structural spacer, which establishes interprotomeric ␣-␣ and ␣- contacts, playing a key role in the oligomerization process
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