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Bruno Curti - One of the best experts on this subject based on the ideXlab platform.

  • Glutamate Synthase: a fascinating pathway from L-glutamine to L-Glutamate.
    Cellular and molecular life sciences : CMLS, 2004
    Co-Authors: R.h.h. Van Den Heuvel, Bruno Curti, Maria A. Vanoni, Andrea Mattevi
    Abstract:

    Glutamate Synthase is a multicomponent iron-sulfur flavoprotein belonging to the class of N-terminal nucleophile amidotransferases. It catalyzes the conversion of L-glutamine and 2-oxoglutarate into two molecules of L-Glutamate. In recent years the X-ray structures of the ferredoxin-dependent Glutamate Synthase and of the a subunit of the NADPH-dependent Glutamate Synthase have become available. Thanks to X-ray crystallography, it is now known that the ammonia reaction intermediate is transferred via an intramolecular tunnel from the amidotransferase domain to the Synthase domain over a distance of about 32A. Although ammonia channeling is a recurrent theme for N-terminal nucleophile and triad-type amidotransferases, the molecular mechanisms of ammonia transfer and its control are different for each known amidotransferase. This review focuses on the intriguing mechanism of action and self-regulation of Glutamate Synthase with a special focus on the structural data.

  • Properties of the recombinant ferredoxin-dependent Glutamate Synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense nadph-dependent enzyme and its isolated α subunit
    Biochemistry, 2002
    Co-Authors: Sergio Ravasio, Bruno Curti, Andrea Mattevi, Laura Dossena, Eugenio Martín-figueroa, Francisco J. Florencio, Paola Morandi, Maria A. Vanoni
    Abstract:

    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.

  • cross talk and ammonia channeling between active centers in the unexpected domain arrangement of Glutamate Synthase
    Structure, 2000
    Co-Authors: Claudia Binda, Bruno Curti, Maria A. Vanoni, Roberto T Bossi, Soichi Wakatsuki, Steffi Arzt, Alessandro Coda, Andrea Mattevi
    Abstract:

    Abstract Introduction: The complex iron-sulfur flavoprotein Glutamate Synthase catalyses the reductive synthesis of L-Glutamate from 2-oxoglutarate and L-glutamine, a reaction in the plant and bacterial pathway for ammonia assimilation. The enzyme functions through three distinct active centers carrying out L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-Glutamate, and electron uptake from an electron donor. Results: The 3.0 A crystal structure of the dimeric 324 kDa core protein of a bacterial Glutamate Synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the asymmetric unit. The 1472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotransferase and FMN binding (β/α) 8 barrel folds, respectively. The other two domains have an unusual "cut (β/α) 8 barrel" topology and an unexpected novel β-helix structure. Channeling of the ammonia intermediate is brought about by an internal tunnel of 31 A length, which runs from the site of L-glutamine hydrolysis to the site of L-Glutamate synthesis. Conclusions: The outstanding property of Glutamate Synthase is the ability to coordinate the activity of its various functional sites to avoid wasteful consumption of L-glutamine. The structure reveals two polypeptide segments that connect the catalytic centers and embed the ammonia tunnel, thus being ideally suited to function in interdomain signaling. Depending on the enzyme redox and ligation states, these signal-transducing elements may affect the active site geometry and control ammonia diffusion through a gating mechanism.

  • Functional properties of recombinant Azospirillum brasilense Glutamate Synthase, a complex iron-sulfur flavoprotein.
    European journal of biochemistry, 2000
    Co-Authors: Helena Stabile, Bruno Curti, Maria A. Vanoni
    Abstract:

    Azospirillum brasilense Glutamate Synthase is a complex iron‐sulfur flavoprotein that catalyses the NADPH-dependent reductive transfer of glutamine amide group to the C(2) carbon of 2-oxoglutarate to yield l-Glutamate. Its catalytically active ab protomer is composed of two dissimilar subunits (a subunit, 164.2 kDa; b subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the b subunit), one FMN (at Site 2, the 2-oxoglutarate/l-Glutamate site in the a subunit) and three different iron‐sulfur clusters (one 3Fe24S center on the a subunit and two 4Fe24S clusters of unknown location). A plasmid harboring the gltD and gltB genes, the genes encoding the Glutamate Synthase b and a subunits, respectively, each one under the control of the T7/lac promoter of pET11a was found to be suitable for the overproduction of Glutamate Synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense Glutamate Synthase could be purified to homogeneity from overproducing E. coli cells by ion exchange chromatography, gel filtration and affinity chromatography on a 2 0 ,5 0 ADP‐Sepharose 4B column. The purified enzyme was indistinguishable from that prepared from Azospirillum cells with respect to cofactor content, N-terminal sequence of the subunits, aggregation state, kinetic and spectroscopic properties. The study of the recombinant holoenzyme allowed us to establish that the tendency of Glutamate Synthase to form a stable (ab)4 tetramer at high protein concentrations is a property unique to the holoenzyme, as the isolated b subunit does not oligomerize, while the isolated Glutamate Synthase a subunit only forms dimers at high protein concentrations. Furthermore, the steady-state kinetic analysis of the Glutamate Synthase reaction was extended to the study of the effect of adenosine-containing nucleotides. Compounds such as cAMP, AMP, ADP and ATP have no effect on the enzyme activity, while the 2 0 -phosphorylated analogs of AMP and NADP(H) analogs act as inhibitors of the reaction, competitive with NADPH. Thus, it can be ruled out that Glutamate Synthase reaction is subjected to allosteric modulation by adenosine containing (di)nucleotides, which may bind to the putative ADP-binding site at the C-terminus of the a subunit. At the same time, the strict requirement of a 2 0 -phosphate group in the pyridine nucleotide for binding to Glutamate Synthase (GltS) was established. Finally, by comparing the inhibition constants exhibited by a series of NADP 1 analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with the effect of substituents on the 3 position of the pyridine ring. With the exception of thio-NADP 1 , which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the interaction between the NADP(H) analog and the enzyme.

  • Glutamate Synthase : A COMPLEX IRON-SULFUR FLAVOPROTEIN
    Cellular and molecular life sciences : CMLS, 1999
    Co-Authors: Maria A. Vanoni, Bruno Curti
    Abstract:

    Glutamate Synthase is a complex iron-sulfur flavoprotein that forms l-Glutamate from l-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of Glutamate Synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of Glutamate Synthase in order to gain insight into the mechanism of the Glutamate Synthase reaction. Sequence analyses also revealed that the small subunit of bacterial Glutamate Synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain.

Maria A. Vanoni - One of the best experts on this subject based on the ideXlab platform.

  • Glutamate Synthase: a fascinating pathway from L-glutamine to L-Glutamate.
    Cellular and molecular life sciences : CMLS, 2004
    Co-Authors: R.h.h. Van Den Heuvel, Bruno Curti, Maria A. Vanoni, Andrea Mattevi
    Abstract:

    Glutamate Synthase is a multicomponent iron-sulfur flavoprotein belonging to the class of N-terminal nucleophile amidotransferases. It catalyzes the conversion of L-glutamine and 2-oxoglutarate into two molecules of L-Glutamate. In recent years the X-ray structures of the ferredoxin-dependent Glutamate Synthase and of the a subunit of the NADPH-dependent Glutamate Synthase have become available. Thanks to X-ray crystallography, it is now known that the ammonia reaction intermediate is transferred via an intramolecular tunnel from the amidotransferase domain to the Synthase domain over a distance of about 32A. Although ammonia channeling is a recurrent theme for N-terminal nucleophile and triad-type amidotransferases, the molecular mechanisms of ammonia transfer and its control are different for each known amidotransferase. This review focuses on the intriguing mechanism of action and self-regulation of Glutamate Synthase with a special focus on the structural data.

  • Properties of the recombinant ferredoxin-dependent Glutamate Synthase of Synechocystis PCC6803. Comparison with the Azospirillum brasilense nadph-dependent enzyme and its isolated α subunit
    Biochemistry, 2002
    Co-Authors: Sergio Ravasio, Bruno Curti, Andrea Mattevi, Laura Dossena, Eugenio Martín-figueroa, Francisco J. Florencio, Paola Morandi, Maria A. Vanoni
    Abstract:

    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.

  • cross talk and ammonia channeling between active centers in the unexpected domain arrangement of Glutamate Synthase
    Structure, 2000
    Co-Authors: Claudia Binda, Bruno Curti, Maria A. Vanoni, Roberto T Bossi, Soichi Wakatsuki, Steffi Arzt, Alessandro Coda, Andrea Mattevi
    Abstract:

    Abstract Introduction: The complex iron-sulfur flavoprotein Glutamate Synthase catalyses the reductive synthesis of L-Glutamate from 2-oxoglutarate and L-glutamine, a reaction in the plant and bacterial pathway for ammonia assimilation. The enzyme functions through three distinct active centers carrying out L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-Glutamate, and electron uptake from an electron donor. Results: The 3.0 A crystal structure of the dimeric 324 kDa core protein of a bacterial Glutamate Synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the asymmetric unit. The 1472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotransferase and FMN binding (β/α) 8 barrel folds, respectively. The other two domains have an unusual "cut (β/α) 8 barrel" topology and an unexpected novel β-helix structure. Channeling of the ammonia intermediate is brought about by an internal tunnel of 31 A length, which runs from the site of L-glutamine hydrolysis to the site of L-Glutamate synthesis. Conclusions: The outstanding property of Glutamate Synthase is the ability to coordinate the activity of its various functional sites to avoid wasteful consumption of L-glutamine. The structure reveals two polypeptide segments that connect the catalytic centers and embed the ammonia tunnel, thus being ideally suited to function in interdomain signaling. Depending on the enzyme redox and ligation states, these signal-transducing elements may affect the active site geometry and control ammonia diffusion through a gating mechanism.

  • Functional properties of recombinant Azospirillum brasilense Glutamate Synthase, a complex iron-sulfur flavoprotein.
    European journal of biochemistry, 2000
    Co-Authors: Helena Stabile, Bruno Curti, Maria A. Vanoni
    Abstract:

    Azospirillum brasilense Glutamate Synthase is a complex iron‐sulfur flavoprotein that catalyses the NADPH-dependent reductive transfer of glutamine amide group to the C(2) carbon of 2-oxoglutarate to yield l-Glutamate. Its catalytically active ab protomer is composed of two dissimilar subunits (a subunit, 164.2 kDa; b subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the b subunit), one FMN (at Site 2, the 2-oxoglutarate/l-Glutamate site in the a subunit) and three different iron‐sulfur clusters (one 3Fe24S center on the a subunit and two 4Fe24S clusters of unknown location). A plasmid harboring the gltD and gltB genes, the genes encoding the Glutamate Synthase b and a subunits, respectively, each one under the control of the T7/lac promoter of pET11a was found to be suitable for the overproduction of Glutamate Synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense Glutamate Synthase could be purified to homogeneity from overproducing E. coli cells by ion exchange chromatography, gel filtration and affinity chromatography on a 2 0 ,5 0 ADP‐Sepharose 4B column. The purified enzyme was indistinguishable from that prepared from Azospirillum cells with respect to cofactor content, N-terminal sequence of the subunits, aggregation state, kinetic and spectroscopic properties. The study of the recombinant holoenzyme allowed us to establish that the tendency of Glutamate Synthase to form a stable (ab)4 tetramer at high protein concentrations is a property unique to the holoenzyme, as the isolated b subunit does not oligomerize, while the isolated Glutamate Synthase a subunit only forms dimers at high protein concentrations. Furthermore, the steady-state kinetic analysis of the Glutamate Synthase reaction was extended to the study of the effect of adenosine-containing nucleotides. Compounds such as cAMP, AMP, ADP and ATP have no effect on the enzyme activity, while the 2 0 -phosphorylated analogs of AMP and NADP(H) analogs act as inhibitors of the reaction, competitive with NADPH. Thus, it can be ruled out that Glutamate Synthase reaction is subjected to allosteric modulation by adenosine containing (di)nucleotides, which may bind to the putative ADP-binding site at the C-terminus of the a subunit. At the same time, the strict requirement of a 2 0 -phosphate group in the pyridine nucleotide for binding to Glutamate Synthase (GltS) was established. Finally, by comparing the inhibition constants exhibited by a series of NADP 1 analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with the effect of substituents on the 3 position of the pyridine ring. With the exception of thio-NADP 1 , which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the interaction between the NADP(H) analog and the enzyme.

  • Glutamate Synthase : A COMPLEX IRON-SULFUR FLAVOPROTEIN
    Cellular and molecular life sciences : CMLS, 1999
    Co-Authors: Maria A. Vanoni, Bruno Curti
    Abstract:

    Glutamate Synthase is a complex iron-sulfur flavoprotein that forms l-Glutamate from l-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of Glutamate Synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of Glutamate Synthase in order to gain insight into the mechanism of the Glutamate Synthase reaction. Sequence analyses also revealed that the small subunit of bacterial Glutamate Synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain.

Ida Brambilla - One of the best experts on this subject based on the ideXlab platform.

David B. Knaff - One of the best experts on this subject based on the ideXlab platform.

  • Glutamate Synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism
    Photosynthesis Research, 2005
    Co-Authors: Akira Suzuki, David B. Knaff
    Abstract:

    Ammonium ion assimilation constitutes a central metabolic pathway in many organisms, and Glutamate Synthase, in concert with glutamine synthetase (GS, EC 6.3.1.2), plays the primary role of ammonium ion incorporation into glutamine and Glutamate. Glutamate Synthase occurs in three forms that can be distinguished based on whether they use NADPH (NADPH-GOGAT, EC 1.4.1.13), NADH (NADH-GOGAT, EC 1.4.1.14) or reduced ferredoxin (Fd-GOGAT, EC 1.4.7.1) as the electron donor for the (two-electron) conversion of L -glutamine plus 2-oxoglutarate to L -Glutamate. The distribution of these three forms of Glutamate Synthase in different tissues is quite specific to the organism in question. Gene structures have been determined for Fd-, NADH- and NADPH-dependent Glutamate Synthases from different organisms, as shown by searches in nucleic acid sequence data banks. Fd-Glutamate Synthase contains two electron-carrying prosthetic groups, the redox properties of which are discussed. A description of the ferredoxin binding by Fd-Glutamate Synthase is also presented. In plants, including nitrogen-fixing legumes, Fd-Glutamate Synthase and NADH-Glutamate Synthase supply Glutamate during the nitrogen assimilation and translocation. The biological functions of Fd-Glutamate Synthase and NADH-Glutamate Synthase, which show a highly tissue-specific distribution pattern, are tightly related to the regulation by the light and metabolite sensing systems. Analysis of mutants and transgenic studies have provided insights into the primary individual functions of Fd-Glutamate Synthase and NADH-Glutamate Synthase. These studies also provided evidence that Glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) does not represent a significant alternate route for Glutamate formation in plants. Taken together, biochemical analysis and genetic and molecular data imply that Fd-Glutamate Synthase incorporates photorespiratory and non-photorespiratory ammonium and provides nitrogen for transport to maintain nitrogen status in plants. Fd-Glutamate Synthase also plays a role that is redundant, in several important aspects, to that played by NADH-Glutamate Synthase in ammonium assimilation and nitrogen transport.

  • The role of lysine and arginine residues at the ferredoxin-binding site of spinach Glutamate Synthase
    Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1993
    Co-Authors: Masakazu Hirasawa, David B. Knaff
    Abstract:

    Treatment of ferredoxin-dependent, spinach Glutamate Synthase with either the arginine-modifying reagent phenylglyoxal or the lysine-modifying reagents N-acetylsuccinimide and dansyl chloride resulted in a significant loss of enzymatic activity when the physiological electron donor, reduced ferredoxin, was used as the electron-donating substrate. In contrast, the reagents caused no inhibition of enzyme activity when the non-physiological reductant, reduced methyl viologen, was used as the electron donor. Formation of an electrostatically-stabilized complex between Glutamate Synthase and ferredoxin prior to exposure of the enzyme to phenylglyoxal or N-acetylsuccinimide protected the enzyme against the loss of ferredoxin-dependent activity caused by either modifying agent. Treatment of Glutamate Synthase with either reagent resulted in a loss of ferredoxin-binding capacity, as assayed by affinity chromatography, gel filtration, spectral perturbations and by the ability of the enzyme to form an active cross-linked complex with ferredoxin. Absorbance and circular dichroism spectra indicated that neither of the modifying reagents produced major conformational changes in the enzyme. These results have been interpreted in terms of a ferredoxin-binding site on Glutamate Synthase, similar to those found on other ferredoxin-dependent chloroplast enzymes, that contains both lysine and arginine residues.

  • The interaction of ferredoxin and Glutamate Synthase: cross-linking and immunological studies.
    Archives of biochemistry and biophysics, 1991
    Co-Authors: Masakazu Hirasawa, Kai-tai Chang, David B. Knaff
    Abstract:

    Abstract The water-soluble carbodiimide, N-ethyl-3-(3-di-methylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, Glutamate Synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to Glutamate Synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:Glutamate Synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on Glutamate Synthase, does not recognize the cross-linked ferredoxin-Glutamate Synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on Glutamate Synthase renders this epitope inaccessible to the antibody.

Remo Reggiani - One of the best experts on this subject based on the ideXlab platform.