Publications by authors named "Maria A Vanoni"

21 Publications

  • Page 1 of 1

Glutamine Synthetase 1 Increases Autophagy Lysosomal Degradation of Mutant Huntingtin Aggregates in Neurons, Ameliorating Motility in a Model for Huntington's Disease.

Cells 2020 01 13;9(1). Epub 2020 Jan 13.

Department of Biosciences, University of Milan, 20133 Milan, Italy.

Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that we associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that we associate with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids' levels that induce a "starvation-like" condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies.
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http://dx.doi.org/10.3390/cells9010196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016901PMC
January 2020

The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties.

Eur Biophys J 2020 Jan 13;49(1):11-19. Epub 2019 Nov 13.

Istituto di Biofisica, Consiglio Nazionale delle Ricerche, via Celoria 26, 20133, Milan, Italy.

Mutations in the gelsolin protein are responsible for a rare conformational disease known as AGel amyloidosis. Four of these mutations are hosted by the second domain of the protein (G2): D187N/Y, G167R and N184K. The impact of the latter has been so far evaluated only by studies on the isolated G2. Here we report the characterization of full-length gelsolin carrying the N184K mutation and compare the findings with those obtained on the wild type and the other variants. The crystallographic structure of the N184K variant in the Ca-free conformation shows remarkable similarities with the wild type protein. Only minimal local rearrangements can be observed and the mutant is as efficient as the wild type in severing filamentous actin. However, the thermal stability of the pathological variant is compromised in the Ca-free conditions. These data suggest that the N to K substitution causes a local disruption of the H-bond network in the core of the G2 domain. Such a subtle rearrangement of the connections does not lead to significant conformational changes but severely affects the stability of the protein.
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http://dx.doi.org/10.1007/s00249-019-01409-9DOI Listing
January 2020

Human MICAL1: Activation by the small GTPase Rab8 and small-angle X-ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8.

Protein Sci 2019 01 31;28(1):150-166. Epub 2018 Oct 31.

Department of Biosciences, University of Milan, Via Celoria 26, 20133, Milan, Italy.

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD-containing monooxygenase-like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C-terminal helical domain presumably shifting the equilibrium from the free - auto-inhibited - conformation to the active one. We here extend the characterization of the MICAL1-Rab8 interaction and show that indeed Rab8, in the active GTP-bound state, stabilizes the active MICAL1 conformation causing a specific four-fold increase of k of the NADPH oxidase reaction. Kinetic data and small-angle X-ray scattering (SAXS) measurements support the formation of a 1:1 complex between full-length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C-terminal Rab-binding domain may mask one of the Rab-binding sites detected with the isolated C-terminal fragment. SAXS-based modeling allowed us to propose the first model of the free full-length MICAL1, which is consistent with an auto-inhibited conformation in which the C-terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.
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http://dx.doi.org/10.1002/pro.3512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295892PMC
January 2019

Cold Denaturation of the HIV-1 Protease Monomer.

Biochemistry 2017 02 13;56(8):1029-1032. Epub 2017 Feb 13.

Structural Biology and NMR Laboratory (SBiNlab), Department of Biology, University of Copenhagen , Ole Maaloees Vej 5, DK-2200 Copenhagen N, Denmark.

The human immunodeficiency virus-1 (HIV-1) protease is a complex protein that in its active form adopts a homodimer dominated by β-sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1 protease that is populated above 0 °C and therefore directly accessible to various spectroscopic approaches. Using nuclear magnetic resonance secondary chemical shifts, temperature coefficients, and protein dynamics, we suggest that the cold-denatured state populates a compact wet globule containing transient non-native-like α-helical elements. From the linearity of the temperature coefficients and the hydrodynamic radii, we propose that the overall architecture of the cold-denatured state is maintained over the temperature range studied.
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http://dx.doi.org/10.1021/acs.biochem.6b01141DOI Listing
February 2017

Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains.

Arch Biochem Biophys 2016 Mar 1;593:24-37. Epub 2016 Feb 1.

Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy. Electronic address:

MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼ 10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 μM) and increases kcat (10-25 s(-1)) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼ 10-fold higher than that of the other forms (3-5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues.
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http://dx.doi.org/10.1016/j.abb.2016.01.016DOI Listing
March 2016

Glutamate synthase: A case-study for in silico drug screening on a complex iron-sulfur flavoenzyme?

Authors:
Maria A Vanoni

Gene 2015 Jun 7;564(2):233-5. Epub 2015 Apr 7.

Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.gene.2015.04.003DOI Listing
June 2015

MICAL, the flavoenzyme participating in cytoskeleton dynamics.

Int J Mol Sci 2013 Mar 27;14(4):6920-59. Epub 2013 Mar 27.

Department of Biosciences, University of Milan, Via Celoria 26, Milano 20133, Italy.

MICAL (from the Molecule Interacting with CasL) indicates a family of recently discovered cytosolic, multidomain proteins, which uniquely couple an N-terminal FAD-containing monooxygenase-like domain to typical calponine homology, LIM and coiled-coil protein-interaction modules. Genetic and cell biology approaches have demonstrated an essential role of the catalytic activity of the monooxygenase-like domain in transducing the signal initiated by semaphorins interaction with their plexin receptors, which results in local actin cytoskeleton disassembly as part of fundamental processes that include differentiation, migration and cell-cell contacts in neuronal and non-neuronal cell types. This review focuses on the structure-function relations of the MICAL monooxygenase-like domain as they are emerging from the available in vitro studies on mouse, human and Drosophila MICAL forms that demonstrated a NADPH-dependent actin depolymerizing activity of MICAL. With Drosophila MICAL forms, actin depolymerization was demonstrated to be associated to conversion of Met44 to methionine sulfone through a postulated hydroxylating reaction. Arguments supporting the concept that MICAL effect on F-actin may be reversible will be discussed.
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http://dx.doi.org/10.3390/ijms14046920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645671PMC
March 2013

Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1.

Arch Biochem Biophys 2011 Nov 16;515(1-2):1-13. Epub 2011 Aug 16.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Universitá degli Studi di Milano, Via Celoria 26, Milan, Italy.

MICALs form a conserved multidomain protein family essential for cytoskeletal rearrangements. To complement structural information available, we produced the FAD-containing monooxygenase-like domain of human MICAL-1 (MICAL-MO) in forms differing for the presence and location of a His-tag, which only influences the protein yields. The K(m) for NADPH of the NADPH oxidase reaction is sensitive to ionic strength and type of ions. The apparent k(cat) (pH 7) is limited by enzyme reduction by NADPH, which occurs without detectable intermediates, as established by anaerobic rapid reaction experiments. The sensitivity to ionic strength and type of ions and the pH dependence of the steady-state kinetic parameters extend MICAL-MO similarity with enzymes of the p-hydroxybenzoate hydroxylase class at the functional level. The reaction is also sensitive to solvent viscosity, providing a tool to monitor the conformational changes predicted to occur during turnover. Finally, it was confirmed that MICAL-MO promotes actin depolymerization, and it was shown that F-actin, but not G-actin, stimulates NADPH oxidation by increasing k(cat) and k(cat)/K(NADPH) (≈5 and ≈200-fold, respectively) with an apparent K(m) for actin of 4.7μM, under conditions that stabilize F-actin. The time-course of NADPH oxidation shows substrate recycling, indicating the possible reversibility of MICAL effect.
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http://dx.doi.org/10.1016/j.abb.2011.08.004DOI Listing
November 2011

L-lactate dehydrogenation in flavocytochrome b2: a first principles molecular dynamics study.

FEBS J 2009 Apr;276(8):2368-80

Dipartimento di Scienze Chimiche ed Ambientali and INSTM, Università dell'Insubria, Como, Italy.

First principles molecular dynamics studies on active-site models of flavocytochrome b2 (L-lactate : cytochrome c oxidoreductase, Fcb2), in complex with the substrate, were carried out for the first time to contribute towards establishing the mechanism of the enzyme-catalyzed L-lactate oxidation reaction, a still-debated issue. In the calculated enzyme-substrate model complex, the L-lactate alpha-OH hydrogen is hydrogen bonded to the active-site base H373 Nepsilon, whereas the Halpha is directed towards flavin N5, suggesting that the reaction is initiated by alpha-OH proton abstraction. Starting from this structure, simulation of L-lactate oxidation led to formation of the reduced enzyme-pyruvate complex by transfer of a hydride from lactate to flavin mononucleotide, without intermediates, but with alpha-OH proton abstraction preceding Halpha transfer and a calculated free energy barrier (12.1 kcal mol(-1)) consistent with that determined experimentally (13.5 kcal mol(-1)). Simulation results also revealed features that are of relevance to the understanding of catalysis in Fcb2 homologs and in a number of flavoenzymes. Namely, they highlighted the role of: (a) the flavin mononucleotide-ribityl chain 2'OH group in maintaining the conserved K349 in a geometry favoring flavin reduction; (b) an active site water molecule belonging to a S371-Wat-D282-H373 hydrogen-bonded chain, conserved in the structures of Fcb2 family members, which modulates the reactivity of the key catalytic histidine; and (c) the flavin C4a-C10a locus in facilitating proton transfer from the substrate to the active-site base, favoring the initial step of the lactate dehydrogenation reaction.
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http://dx.doi.org/10.1111/j.1742-4658.2009.06969.xDOI Listing
April 2009

Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl-tRNA synthetase and determination of its oligomeric structure in solution.

FEBS J 2009 Mar;276(5):1398-417

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milan, Italy.

Mycobacterium tuberculosis glutamyl-tRNA synthetase (Mt-GluRS), encoded by Rv2992c, was overproduced in Escherichia coli cells, and purified to homogeneity. It was found to be similar to the other well-characterized GluRS, especially the E. coli enzyme, with respect to the requirement for bound tRNA(Glu) to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k(cat) (130 min(-1)) and K(M) for tRNA (0.7 microm) and ATP (78 microm), but to differ by a one order of magnitude higher K(M) value for L-Glu (2.7 mm). At variance with the E. coli enzyme, among the several compounds tested as inhibitors, only pyrophosphate and the glutamyl-AMP analog glutamol-AMP were effective, with K(i) values in the mum range. The observed inhibition patterns are consistent with a random binding of ATP and L-Glu to the enzyme-tRNA complex. Mt-GluRS, which is predicted by genome analysis to be of the non-discriminating type, was not toxic when overproduced in E. coli cells indicating that it does not catalyse the mischarging of E. coli tRNA(Gln) with L-Glu and that GluRS/tRNA(Gln) recognition is species specific. Mt-GluRS was significantly more sensitive than the E. coli form to tryptic and chymotryptic limited proteolysis. For both enzymes chymotrypsin-sensitive sites were found in the predicted tRNA stem contact domain next to the ATP binding site. Mt-GluRS, but not Ec-GluRS, was fully protected from proteolysis by ATP and glutamol-AMP. Small-angle X-ray scattering showed that, at variance with the E. coli enzyme that is strictly monomeric, the Mt-GluRS monomer is present in solution in equilibrium with the homodimer. The monomer prevails at low protein concentrations and is stabilized by ATP but not by glutamol-AMP. Inspection of small-angle X-ray scattering-based models of Mt-GluRS reveals that both the monomer and the dimer are catalytically active. By using affinity chromatography and His(6)-tagged forms of either GluRS or glutamyl-tRNA reductase as the bait it was shown that the M. tuberculosis proteins can form a complex, which may control the flux of Glu-tRNA(Glu) toward protein or tetrapyrrole biosynthesis.
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http://dx.doi.org/10.1111/j.1742-4658.2009.06880.xDOI Listing
March 2009

The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications.

J Biol Chem 2008 Mar 16;283(13):8237-49. Epub 2008 Jan 16.

Département de Biologie Structurale, IMPMC-UMR 7590, CNRS, Universités Paris 6 et Paris 7, IPGP, Paris, France.

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.
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http://dx.doi.org/10.1074/jbc.M708529200DOI Listing
March 2008

Does negative hyperconjugation assist enzymatic dehydrogenations?

Chemphyschem 2007 Jun;8(9):1283-8

Dipartimento di Scienze Chimiche ed Ambientali and INSTM, Università degli Studi dell'Insubria, Via Lucini 3, I-22100 Como, Italy.

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http://dx.doi.org/10.1002/cphc.200700085DOI Listing
June 2007

Activation and coupling of the glutaminase and synthase reaction of glutamate synthase is mediated by E1013 of the ferredoxin-dependent enzyme, belonging to loop 4 of the synthase domain.

Biochemistry 2007 Apr 21;46(15):4473-85. Epub 2007 Mar 21.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.

Crystal structures of glutamate synthase suggested that a conserved glutamyl residue of the synthase domain (E1013 of Synechocystis sp. PCC 6803 ferredoxin-dependent glutamate synthase, FdGltS) may play a key role in activating glutamine binding and hydrolysis and ammonia transfer to the synthase site in this amidotransferase, in response to the ligation and redox state of the synthase site. The E1013D, N, and A, variants of FdGltS were overproduced in Escherichia coli cells, purified, and characterized. The amino acyl substitutions had no effect on the reactivity of the synthase site nor on the interaction with ferredoxin. On the contrary, a dramatic decrease of activity was observed with the D (approximately 100-fold), N and A (approximately 10,000-fold) variants, mainly due to an effect on the maximum velocity of the reaction. The E1013D variant showed coupling between glutamine hydrolysis at the glutaminase site and 2-oxoglutarate-dependent L-glutamate synthesis at the synthase site, but a sigmoid dependence of initial velocity on L-glutamine concentration. The E1013N variant exhibited hyperbolic kinetics, but the velocity of glutamine hydrolysis was twice that of glutamate synthesis from 2-oxoglutarate at the synthase site. These results are consistent with the proposed role of E1013 in signaling the presence of 2-oxoglutarate (and reducing equivalents) at the synthase site to the glutaminase site in order to activate it and to promote ammonia transfer to the synthase site through the ammonia tunnel. The sigmoid dependence of the initial velocity of the glutamate synthase reaction of the E1013D mutant on glutamine concentration provides evidence for a participation of glutamine in the activation of glutamate synthase during the catalytic cycle.
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http://dx.doi.org/10.1021/bi061865dDOI Listing
April 2007

Structure-function studies on the complex iron-sulfur flavoprotein glutamate synthase: the key enzyme of ammonia assimilation.

Photosynth Res 2005 ;83(2):219-38

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, Milan 20131, Italy.

Glutamate synthases are complex iron-sulfur flavoproteins that participate in the essential ammonia assimilation pathway in microorganisms and plants. The recent determination of the 3-dimensional structures of the alpha subunit of the NADPH-dependent glutamate synthase form and of the ferredoxin-dependent enzyme of Synechocystis sp. PCC 6803 provides a framework for the interpretation of the functional properties of these enzymes, and highlights protein segments most likely involved in control and coordination of the partial catalytic activities of glutamate synthases, which take place at sites distant from each other in space. In this review, we focus on the current knowledge on structure-function relationships in glutamate synthases, and we discuss open questions on the mechanisms of control of the enzyme reaction and of electron transfer among the enzyme flavin cofactors and iron-sulfur clusters.
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http://dx.doi.org/10.1007/s11120-004-2438-zDOI Listing
September 2005

The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit.

Arch Biochem Biophys 2005 Apr;436(2):355-66

Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active alphabeta protomer (alpha subunit, 162kDa; beta subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S](0,+1), and two [4Fe-4S](+1,+2) clusters. The structure of the alpha subunit has been determined providing information on the mechanism of ammonia transfer from L-glutamine to 2-oxoglutarate through a 30 A-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS beta subunit with alanyl residues. The engineered genes encoding the beta subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type alpha subunit gene. In all cases the C/A substitutions prevented alpha and beta subunits association to yield the GltS alphabeta protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S](+1,+2) clusters within the N-terminal region of the beta subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in alphabeta heterodimer formation by structuring an N-terminal [Fe-S] beta subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal beta subunit-like domain.
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http://dx.doi.org/10.1016/j.abb.2005.02.009DOI Listing
April 2005

Structure--function studies on the iron-sulfur flavoenzyme glutamate synthase: an unexpectedly complex self-regulated enzyme.

Arch Biochem Biophys 2005 Jan;433(1):193-211

Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita' degli Studi di Milano, Via Celoria 26, 20131 Milan, Italy.

Glutamate synthase (GltS) is, with glutamine synthetase, the key enzyme of ammonia assimilation in bacteria, microorganisms and plants. GltS isoforms result from the assembly and co-evolution of conserved functional domains. They share a common mechanism of reductive glutamine-dependent glutamate synthesis from 2-oxoglutarate, which takes place within the alpha subunit ( approximately 150 kDa) of the NADPH-dependent bacterial enzyme and the corresponding polypeptides of other GltS forms, and involves: (i) an Ntn-type amidotransferase domain and (ii) a flavin mononucleotide-containing (beta/alpha)(8) barrel synthase domain connected by (iii) a approximately 30 A-long intramolecular ammonia tunnel. The synthase domain harbors the [3Fe/4S](0,+1) cluster of the enzyme, which participates in the electron transfer process from the physiological reductant: reduced ferredoxin in the plant-type enzyme or NAD(P)H in the bacterial and the non-photosynthetic eukaryotic form. The NAD(P)H-dependent GltS requires a tightly bound flavin adenine dinucleotide-dependent reductase (beta subunit, approximately 50 kDa), also determining the presence of two low-potential [4Fe-4S](+1,+2) clusters. Structural, functional and computational data available on GltS and related enzymes show how the enzyme may control and coordinate the reactions taking place at the glutaminase and synthase sites by sensing substrate binding and cofactor redox state.
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http://dx.doi.org/10.1016/j.abb.2004.08.033DOI Listing
January 2005

Molecular dynamics simulation of the interaction between the complex iron-sulfur flavoprotein glutamate synthase and its substrates.

Protein Sci 2004 Nov;13(11):2979-91

Department of Chemistry, University of Rome La Sapienza, Piazzale Aldo Moro 5, Rome 00185, Italy.

Glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of L-glutamine amide group to the C2 carbon of 2-oxoglutarate yielding two molecules of L-glutamate. Molecular dynamics calculations in explicit solvent were carried out to gain insight into the conformational flexibility of GltS and into the role played by the enzyme substrates in regulating the catalytic cycle. We have modelled the free (unliganded) form of Azospirillum brasilense GltS alpha subunit and the structure of the reduced enzyme in complex with the L-glutamine and 2-oxoglutarate substrates starting from the crystallographically determined coordinates of the GltS alpha subunit in complex with L-methionine sulphone and 2-oxoglutarate. The present 4-ns molecular dynamics calculations reveal that the GltS glutaminase site may exist in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Substrates binding also induce (1) closure of the loop formed by residues 263-271 with partial shielding of the glutaminase site from solvent, and (2) widening of the ammonia tunnel entrance at the glutaminase end to allow for ammonia diffusion toward the synthase site. The Q-loop of glutamate synthase, which acts as an active site lid in other amidotransferases, seems to maintain an open conformation. Finally, binding of L-methionine sulfone, a glutamine analog that mimics the tetrahedral transient species occurring during its hydrolysis, causes a coordinated rigid-body motion of segments of the glutaminase domain that results in the inactive conformation observed in the crystal structure of GltS alpha subunit.
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http://dx.doi.org/10.1110/ps.04863104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2286594PMC
November 2004

The active conformation of glutamate synthase and its binding to ferredoxin.

J Mol Biol 2003 Jun;330(1):113-28

Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207, Italy.

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.
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http://dx.doi.org/10.1016/s0022-2836(03)00522-9DOI Listing
June 2003

Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering.

J Biol Chem 2003 Aug 30;278(32):29933-9. Epub 2003 May 30.

European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany.

Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alpha beta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alpha beta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of alpha subunits in the (alpha beta)4 holoenzyme is similar to that in the tetrameric alpha 4 complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha 4 was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alpha beta holoenzyme, a putative alpha beta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha 4, but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alpha beta protomers, which is consistent with the available biochemical evidence.
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http://dx.doi.org/10.1074/jbc.M304147200DOI Listing
August 2003

Properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense NADPH-dependent enzyme and its isolated alpha subunit.

Biochemistry 2002 Jun;41(25):8120-33

Dipartimento di Fisiologia e Biochimica Generali, Universita' di Milano, Via Celoria 26, 20131 Milan, Italy.

The properties of the recombinant ferredoxin-dependent glutamate synthase of Synechocystis PCC6803 were determined by means of kinetic and spectroscopic approaches in comparison to those exhibited by the bacterial NADPH-dependent enzyme form. The ferredoxin-dependent enzyme was found to be similar to the bacterial glutamate synthase alpha subunit with respect to cofactor content (one FMN cofactor and one [3Fe-4S] cluster per enzyme subunit), overall absorbance properties, and reactivity of the FMN N(5) position with sulfite, as expected from the similar primary structure of ferredoxin-dependent glutamate synthase and of the bacterial NADPH-dependent glutamate synthase alpha subunit. The ferredoxin- and NADPH-dependent enzymes were found to differ with respect to the apparent midpoint potential values of the FMN cofactor and of the [3Fe-4S] cluster, which are less negative in the ferredoxin-dependent enzyme form. This feature is, at least in part, responsible for the efficient oxidation of L-glutamate catalyzed by this enzyme form, but not by the bacterial NADPH-dependent counterpart. At variance with earlier reports on ferredoxin-dependent glutamate synthase, in the Synechocystis enzyme the [3Fe-4S] cluster is not equipotential with the flavin cofactor. The present studies also demonstrated that binding of reduced ferredoxin to ferredoxin-dependent glutamate synthase is essential in order to activate reaction steps such as glutamine binding, hydrolysis, or ammonia transfer from the glutamine amidotransferase site to the glutamate synthase site of the enzyme. Thus, ferredoxin-dependent glutamate synthase seems to control and coordinate catalytic activities taking place at its subsites by regulating the reactions of the glutamine amidotransferase site. Association with reduced ferredoxin appears to be necessary, but not sufficient, to trigger the required activating conformational changes.
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http://dx.doi.org/10.1021/bi020083rDOI Listing
June 2002

Structural studies on the synchronization of catalytic centers in glutamate synthase.

J Biol Chem 2002 Jul 19;277(27):24579-83. Epub 2002 Apr 19.

Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207, 27100 Pavia, Italy.

The complex iron-sulfur flavoprotein glutamate synthase (GltS) plays a prominent role in ammonia assimilation in bacteria, yeasts, and plants. GltS catalyzes the formation of two molecules of l-glutamate from 2-oxoglutarate and l-glutamine via intramolecular channeling of ammonia. GltS has the impressive ability of synchronizing its distinct catalytic centers to avoid wasteful consumption of l-glutamine. We have determined the crystal structure of the ferredoxin-dependent GltS in several ligation and redox states. The structures reveal the crucial elements in the synchronization between the glutaminase site and the 2-iminoglutarate reduction site. The structural data combined with the catalytic properties of GltS indicate that binding of ferredoxin and 2-oxoglutarate to the FMN-binding domain of GltS induce a conformational change in the loop connecting the two catalytic centers. The rearrangement induces a shift in the catalytic elements of the amidotransferase domain, such that it becomes activated. This machinery, over a distance of more than 30 A, controls the ability of the enzyme to bind and hydrolyze the ammonia-donating substrate l-glutamine.
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http://dx.doi.org/10.1074/jbc.M202541200DOI Listing
July 2002