Quaternary Structure of Azospirillum brasilense NADPH-dependent Glutamate Synthase in Solution as Revealed by Synchrotron Radiation X-ray Scattering

Petoukhov, Maxim V.; Svergun, Dmitri I.; Konarev, Petr V.; Ravasio, Sergio; Heuvel, Robert H. H. van den; Curti, Bruno; Vanoni, Maria A.

doi:10.1074/jbc.m304147200

Cited by 23 publications

(19 citation statements)

References 31 publications

(56 reference statements)

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“…A form of NmFNR, with spectral properties distinct from both the 4Fe-4S cluster and the 2Fe-2S cluster, forms over the same time scale as oxygen utilization occurs. (23)(24)(25).…”

Section: Discussionmentioning

confidence: 99%

Binding to DNA Protects Neisseria meningitidis Fumarate and Nitrate Reductase Regulator (FNR) from Oxygen

Edwards

Cole

Green

et al. 2010

Journal of Biological Chemistry

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Here, we report the overexpression, purification, and characterization of the transcriptional activator fumarate and nitrate reductase regulator from the pathogenic bacterium Neisseria meningitidis (NmFNR). Like its homologue from Escherichia coli (EcFNR), NmFNR binds a 4Fe-4S cluster, which breaks down in the presence of oxygen to a 2Fe-2S cluster and subsequently to apo-FNR. The kinetics of NmFNR cluster disassembly in the presence of oxygen are 2-3؋ slower than those previously reported for wild-type EcFNR, but similar to constitutively active EcFNR* mutants, consistent with earlier work in which we reported that the activity of FNR-dependent promoters in N. meningitidis

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“…A form of NmFNR, with spectral properties distinct from both the 4Fe-4S cluster and the 2Fe-2S cluster, forms over the same time scale as oxygen utilization occurs. (23)(24)(25).…”

Section: Discussionmentioning

confidence: 99%

Binding to DNA Protects Neisseria meningitidis Fumarate and Nitrate Reductase Regulator (FNR) from Oxygen

Edwards

Cole

Green

et al. 2010

Journal of Biological Chemistry

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“…In Ref. 54, both gel filtration and SAXS data pointed to a tetrameric assembly, and a P222 symmetry was adopted for the reconstruction. The differences between the "old" and "new" SAXS models in supplemental Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The addition of salt led to dissociation of ␣ 6 hexamers similar to that observed with GltS, but at 2 mg/ml and in the presence of 1 M NaCl, ␣ 6 hexamers were almost fully dissociated into ␣ monomers (supplemental Table 3). The heterogeneity of the previous GltS preparation led to underestimating the oligomer stoichiometry (54). Both gel filtration and SAXS data pointed to a tetrameric assembly (the former due to the nonlinearity of calibration at high MM, the latter due to partial dissociation).…”

Section: Saxs and Stoichiometry Of The Nadph-glts Complex In Solutionmentioning

confidence: 99%

“…Earlier SAXS results suggested a tetrameric (␣␤) 4 assembly of GltS in solution (54), assuming monodispersity of the GltS samples. Given the new evidence for the co-existence of different GltS oligomers, the previous data were reanalyzed, leading us to the conclusion that the samples studied at that time were actually mixtures of about 20% ␣␤ protomers and 80% (␣␤) 6 hexamers (supplemental Fig.…”

Section: Saxs and Stoichiometry Of The Nadph-glts Complex In Solutionmentioning

confidence: 99%

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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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“…Hammel et al (2002)], bovine serum albumin [e.g. Petoukhov et al (2003)] or glucose isomerase (Kozak, 2005). Alternatively, scattering from secondary standards like Lupolen (Kratky, 1964), or water (Orthaber et al, 2000) can be used to obtain the scattering from the solute on the absolute scale and then to calculate the MM.…”

Section: Introductionmentioning

confidence: 99%

Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering

2007

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One of the most important overall parameters, which can be derived from small‐angle X‐ray scattering (SAXS) experiments on macromolecular solutions is the molecular mass (MM) of the solute. In particular, for a monodisperse protein solution, MM of the solute is calculated from the extrapolated scattering intensity at zero angle I(0). Assessing MM by SAXS provides valuable information about the oligomeric state and absence of unspecific aggregation in solution. The value of MM can either be estimated by comparison with a protein standard with a known MM or by determining the absolute scattering intensity using, e.g., water scattering. In both cases, knowledge about the solute concentration and about the partial specific volume of the protein is required. By measuring 13 well characterized globular proteins with MMs ranging from 13.7 to 669 kDa we analyze the sources of possible systematic deviations and assess the accuracy of MM determination using SAXS. The data indicate that all these proteins have approximately the same `effective' value of the partial specific volume of about 0.7425 cm3 g−1. It is shown that both inter‐protein and water calibration can be used for molecular mass determination by SAXS and in most cases the errors do not exceed 10%.

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Quaternary Structure of Azospirillum brasilense NADPH-dependent Glutamate Synthase in Solution as Revealed by Synchrotron Radiation X-ray Scattering

Cited by 23 publications

References 31 publications

Binding to DNA Protects Neisseria meningitidis Fumarate and Nitrate Reductase Regulator (FNR) from Oxygen

Binding to DNA Protects Neisseria meningitidis Fumarate and Nitrate Reductase Regulator (FNR) from Oxygen

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

Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering

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