Glutamate synthase: a complex iron-sulfur flavoprotein

Vanoni, Maria A.; Curti, Bruno

doi:10.1007/s000180050319

Cited by 110 publications

(109 citation statements)

References 43 publications

(93 reference statements)

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“…It has been previously shown (1,12,14) that ␤GltS and the N-terminal region of DPD are similar to each other. Thus, the DPD atomic coordinates (Protein Data Bank code 1h7w, chain A, residues 31-520) were used as a structural template to produce a homology model of ␤GltS ( Fig.…”

Section: Homology Modeling Of the Glts ␤ Subunitmentioning

confidence: 84%

“…Furthermore, the location and the role of the [4Fe-4S] 1ϩ/2ϩ clusters of ␤GltS only rely on indirect observations. Interestingly, the sequence of ␤GltS and, in particular, the spacing of its N-terminal conserved cysteine residues are similar to those of several other proteins or protein domains (1), among which only the Pyrococcus furiosus sulfide dehydrogenase ␣ subunit (11) and the bovine dihydropyrimidine dehydrogenase (DPD) (12)(13)(14) have been characterized to different extents. Site-directed mutagenesis experiments allowed us to confirm the location of the DPD-like [4Fe-4S] clusters of GltS within ␤GltS.…”

mentioning

confidence: 87%

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

Cottevieille

Larquet

Jonić

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Homology Modeling Of the Glts ␤ Subunitmentioning

confidence: 84%

mentioning

confidence: 87%

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

Cottevieille

Larquet

Jonić

et al. 2008

Journal of Biological Chemistry

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“…DsrL matches the conserved GltD domain (␤-subunit of glutamate synthases), which transfers electrons from NAD(P)H to an acceptor protein or protein domain (73). DsrL contains the adenylate binding motif GXGXXG/A/P (77) twice; the first probably interacts with the adenylate portion of flavin adenine dinucleotide (FAD), and the second interacts with the pyridine nucleotide cofactor (54).…”

Section: Resultsmentioning

confidence: 99%

Novel Genes of the dsr Gene Cluster and Evidence for Close Interaction of Dsr Proteins during Sulfur Oxidation in the Phototrophic Sulfur Bacterium Allochromatium vinosum

et al. 2005

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Seven new genes designated dsrLJOPNSR were identified immediately downstream of dsrABEFHCMK, completing the dsr gene cluster of the phototrophic sulfur bacterium Allochromatium vinosum D (DSM 180 T ). Interposon mutagenesis proved an essential role of the encoded proteins for the oxidation of intracellular sulfur, an obligate intermediate during the oxidation of sulfide and thiosulfate. While dsrR and dsrS encode cytoplasmic proteins of unknown function, the other genes encode a predicted NADPH:acceptor oxidoreductase (DsrL), a triheme c-type cytochrome (DsrJ), a periplasmic iron-sulfur protein (DsrO), and an integral membrane protein (DsrP). DsrN resembles cobyrinic acid a,c-diamide synthases and is probably involved in the biosynthesis of siro(heme)amide, the prosthetic group of the dsrAB-encoded sulfite reductase. The presence of most predicted Dsr proteins in A. vinosum was verified by Western blot analysis. With the exception of the constitutively present DsrC, the formation of Dsr gene products was greatly enhanced by sulfide. DsrEFH were purified from the soluble fraction and constitute a soluble ␣ 2 ␤ 2 ␥ 2 -structured 75-kDa holoprotein. DsrKJO were purified from membranes pointing at the presence of a transmembrane electron-transporting complex consisting of DsrKMJOP. In accordance with the suggestion that related complexes from dissimilatory sulfate reducers transfer electrons to sulfite reductase, the A. vinosum Dsr complex is copurified with sulfite reductase, DsrEFH, and DsrC. We therefore now have an ideal and unique possibility to study the interaction of sulfite reductase with other proteins and to clarify the long-standing problem of electron transport from and to sulfite reductase, not only in phototrophic bacteria but also in sulfate-reducing prokaryotes.Phototrophic purple and green sulfur-oxidizing bacteria use sulfur compounds as electron donors for reductive carbon dioxide fixation during photolithotrophic growth (7, 10). In these organisms, light energy is used to transfer electrons from sulfur compounds to the level of the more highly reducing electron carriers NAD(P) ϩ and ferredoxin. In our laboratory we seek to understand oxidative sulfur metabolism in anoxygenic phototrophic bacteria by using the genetically accessible ␥-proteobacterium Allochromatium vinosum (formerly Chromatium vinosum [34]) as our model organism. It is a purple sulfur bacterium belonging to the family Chromatiaceae. A. vinosum carries out the complete eight-electron oxidations of sulfide and thiosulfate to sulfate. Intracellularly stored sulfur globules are an obligate intermediate in the process (57). The sulfur in the globules is present in the molecular structure of sulfur chains (58) and is enclosed by a protein envelope, a feature shared by most if not all of the chemotrophic sulfur-oxidizing bacteria that form intracellular sulfur globules (9, 14, 51). Topologically, the sulfur globules of A. vinosum and probably of other members of the Chromatiaceae are located extracytoplasmically, in the periplasm (51).O...

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“…1). This enzyme with a monomeric mass of 165 kDa is of extreme importance for ammonia assimilation in plants and bacteria, where it catalyzes the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate (Vanoni & Curti, 1999). The enzyme contains a flavin mononucleotide (FMN) and a 3Fe-4S cluster as non-covalently bound cofactors.…”

Section: Electrospray Ionization Of Biomacromoleculesmentioning

confidence: 99%

Investigation of intact protein complexes by mass spectrometry

Heck

Heuvel

2004

Mass Spectrometry Reviews

553

554

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I. Introduction 00 II. Electrospray Ionization of Biomacromolecules 00 III. Mass Analyzers for Biomacromolecular Mass Spectrometry 00 A. Collisional Cooling and/or Focusing 00 B. Alternative Mass Analyzers 00 IV. Solution‐Phase Properties of Proteins Complexes 00 A. Protein–Protein Interactions 00 B. Cooperativity and Cofactors 00 C. Dynamics of Assembly 00 V. Gas‐Phase Properties of Protein Complexes 00 A. Tandem Mass Spectrometry 00 B. Charge Separation in Protein Dissociation 00 VI. Future Outlook 00 Acknowledgments 00 References 00 Mass spectrometry has grown in recent years to a well‐accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non‐covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non‐covalent protein–protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights. © 2004 Wiley Periodicals, Inc., Mass Spec Rev

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Glutamate synthase: a complex iron-sulfur flavoprotein

Cited by 110 publications

References 43 publications

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

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

Novel Genes of the dsr Gene Cluster and Evidence for Close Interaction of Dsr Proteins during Sulfur Oxidation in the Phototrophic Sulfur Bacterium Allochromatium vinosum

Investigation of intact protein complexes by mass spectrometry

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