Glutamate synthase: a fascinating pathway from L-glutamine to L-glutamate

Heuvel, Robert H. H. van den; Curti, Bruno; Vanoni, Maria A.; Mattevi, Andrea

doi:10.1007/s00018-003-3316-0

Cited by 80 publications

(51 citation statements)

References 94 publications

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“…There appeared to be an increased flow of glutamate to ␣-keto-glutarate, as indicated by the up-regulation of the serine biosynthesis pathway. In addition, glutamate synthase contains an iron-sulfur cluster and has close structural homology with glutamine phosphoribosylpyrophosphate amidotransferase (71). This latter enzyme from B. subtilis is known to be inactivated by O 2 in stationary phase (9).…”

Section: Resultsmentioning

confidence: 99%

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

et al. 2005

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The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low-nutrient environments, a characteristic of most heavy metal-contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well-developed genetics. We have identified the pathways responding to heavy-metal toxicity in C. crescentus to provide insights for the possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium, and uranium) and analyzed genome-wide transcriptional activities postexposure using an Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be the formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase sodA). Glutathione S-transferase, thioredoxin, glutaredoxins, and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium, while a sulfate transporter was down-regulated to reduce nonspecific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome by using whole-genome microarrays.Potentially hazardous levels of heavy metals have dispersed into subsurface sediment and groundwater in a number of metal-contaminated sites and represent a challenge for environmental restoration. Effective bioremediation of these sites requires knowledge of genetic pathways for resistance and biotransformation by component organisms within a microbial community. However, a comprehensive understanding of bacterial mechanisms of heavy metal toxicity and resistance has yet to be achieved. While many metals are essential to microbial function, heavy metals, i.e., most of those with a density above 5 g/cm 3 , have toxic effects on cellular metabolism (46). The majority of heavy metals are transition elements with incompletely filled d orbitals providing heavy metal cations which can form complex compounds with redox activity (46,70). Therefore, it is important to the health of the organism that the intracellular concentrations of heavy metal ions are tightly controlled. However, due to their structural and valence similarities to nontoxic metals, heavy metals are often transported into the cytoplasm through constitutively expressed nonspecific transport systems (46). As such, heavy metals invariably find their way into the cell. Once inside the cell, toxic effects of heavy metals...

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Section: Resultsmentioning

confidence: 99%

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

et al. 2005

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“…2) and the estimates of their midpoint potential (E m ) values (supplemental Table 5) (53,58) allow us to propose that electron transfer from FAD to FMN takes place with two one-electron transfer processes that follow the same pathway. However, the energetics of transfer of the first (empty circle, continuous arrows, steps [2][3][4] and the second electron (full circle, dashed arrows, steps 5-7) differ due to the different E m values of oxidized/semiquinone and semiquinone/hydroquinone forms of both flavin cofactors. In these schemes, the two low potential [4Fe-4S] clusters of GltS (53) are assumed to be equipotential with E m values in the range of that of the NADP ϩ /NADPH couple (Ϫ340 mV).…”

Section: Discussionmentioning

confidence: 99%

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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“…Because all ATP pyrophosphatases convert ATP to AMP, the C-terminal active site of ASNS likely catalyzes activation of the side-chain carboxylate of aspartate to form an electrophilic intermediate, β-aspartyl-AMP (βAspAMP) 1, and inorganic pyrophosphate (PP i ) (Scheme 2) (28,55). As observed in other glutaminedependent amidotransferases (44,46,(56)(57)(58)(59)(60)(61), the two active sites of AS-B are linked by a solvent-inaccessible, intramolecular "tunnel" that is sufficiently wide to allow passage of an ammonia molecule (Figure 1b) (62). Glutamine-dependent asparagine production is therefore accomplished using ammonia as a common intermediate to couple the two "halfreactions" carried out in the independent active sites of the enzyme.…”

Section: Structure Of Asparagine Synthetasementioning

confidence: 96%

Asparagine Synthetase Chemotherapy

Richards

Kilberg

2006

Annu. Rev. Biochem.

204

221

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Modern clinical treatments of childhood acute lymphoblastic leukemia (ALL) employ enzymebased methods for depletion of blood asparagine in combination with standard chemotherapeutic agents. Significant side effects can arise in these protocols and, in many cases, patients develop drug-resistant forms of the disease that may be correlated with up-regulation of the enzyme glutamine-dependent asparagine synthetase (ASNS). Though the precise molecular mechanisms that result in the appearance of drug resistance are the subject of active study, potent ASNS inhibitors may have clinical utility in treating asparaginase-resistant forms of childhood ALL. This review provides an overview of recent developments in our understanding of (a) the structure and catalytic mechanism of ASNS, and (b) the role that ASNS may play in the onset of drug-resistant childhood ALL. In addition, the first successful, mechanism-based efforts to prepare and characterize nanomolar ASNS inhibitors are discussed, together with the implications of these studies for future efforts to develop useful drugs.

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Glutamate synthase: a fascinating pathway from L-glutamine to L-glutamate

Cited by 80 publications

References 94 publications

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

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

Asparagine Synthetase Chemotherapy

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