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| Maria Antonietta Vanoni - Glutamate Synthase, a Complex Iron-Sulfur Flavoprotein: the Quest for Its Three-Dimensional Structure | |
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Maria Antonietta Vanoni
Dipartimento di Scienze Biomolecolari e Biotecnologie Universita? degli Studi di Milano, Italy
The lecture will be divided in two parts and will address the current status of our understanding of the structure-function relationships in glutamate synthases (GltS) and, if time allows, glutamyl-tRNA synthetase (GluRS) as gathered from the combined approaches of X-ray crystallography, small-angle X-ray scattering, cryo electron microscopy , atomic force microscopy and, mainly, kinetic and spectroscopic studies on these enzymes.
Glutamate synthase is a complex iron sulfur flavoprotein that catalyses the reductive synthesis of L-Glu form L-Gln and 2-oxoglutarate (2-OG). It is responsible with glutamine synthetase of ammonia assimilation processes in microorganisms (including bacteria used as biofertilisers and pathogens) and plants. The enzyme is only partially conserved in animals where it is however found in the model organism C. elegans, in Plasmodia, Toxoplasma and in the plasmodium carrier Anopheles gambiae. We are studying the Azospirillum brasilense NADPH-GltS, typical of eubacteria and a valid model of the NADH-dependent eukaryotic enzyme, and the plant-type cyanobacterial Fd-dependent GltS.
With its 5 different redox centers that form an intramolecular electron transport chain, the NADPH-GltS is a model for other enzymes containing multiple redox centers. With the growing class of amidotransferases all GltS share the ability to hydrolyse glutamine and to channel it to the acceptor (2-OG) site through a 30 A-long intramolecular tunnel with tight control and coordination of ammonia release and production of the aminated product , which occur at distinct active sites separated by the tunnel. Reversible interaction between Fd and Fd-GltS also has a clear activating role on the glutamate synthase reaction.
In order to correlate the functional (kinetic, mechanistic, spectroscopic) studies that are being carried out in our laboratory on wild-type and mutant forms of the enzyme with its structural features we undertook crystallographic experiments, which however led to the determination of the high resolution structure of only the α subunit (~150 kDa) of the NADPH-dependent enzyme upon loss of the β subunit (~50 kDa) during crystallization experiments. In order to establish whether the enzyme is conformationally heterogeneous in solution, which are the factors that may stabilize one or the other prevailing conformation we are using a combination of small-angle X-ray scattering, cryoEM and atomic force microscopy , which are flanked by site-directed mutagenesis and kinetic and spctrocopic studies.
The data we obtained will be presented and discussed to highlight how all these approaches may be used synergistically to establish the quaternary structure of the enzyme. It is anticipated that the enzyme appears heterogeneous in solution and we are currently evaluating if the observed heterogeneity is a preparation artifact or if it relates to conformational changes induced by ligand binding giving rise to alternative quaternary assemblies as proposed for morpheeins.
Glutamyl-trNA synthetase of Mycobacterium tuberculosis (Mt-GluRS) is essential for both protein and tetrapyrrol biosynthsis, thus an ideal target for the design of novel antitubercular drugs. We are studying the functional and structural properties of Mt-GluRS in comparison with the functionally well-characterised E. coli enzyme and the Thermus thermophilus and Thermosynechococcus elongatus enzymes for which high resolution structures are available. Crystallyzation experiments suggested the the enzyme is conformationally heterogeneous, leading us to use SAXS to evaluate the extent of such a heterogeneity and which may be the factors that induce stabilization of one of the forms. We will discuss the results of the experiments in light of complementary kinetic, mechanistic and spectroscopic studies being carried out in the laboratory.
Glutamate synthase
Van den Heuvel, R.H.H., Curti, B, Vanoni, M.A. and Mattevi, A. (2004) Glutamate synthase: a fascinating pathway from L-glutamine to L-glutamate Cell. Mol. Life Sci. 61, 669-681.
Vanoni, M.A., Dossena, L., van den Heuvel, R.H.H. and Curti, B. (2005) Structure-function studies on the complex iron-sulfur flavoprotein glutamate synthase: the key enzyme of ammonia assimilation. Photosynthesis research 83, 219-238.
Vanoni, M.A. and Curti, B. (2005) Structure-function studies on the iron-sulfur flavoenzyme glutamate synthase: an unexpectedly complex self-regulated enzyme. Arch. Biochem. Biophys. 433, 193-211.
Morpheeins
Jaffe EK. Morpheeins--a new structural paradigm for allosteric regulation. (2005) Trends Biochem Sci. 30, 490-497.
Glutamyl-tRNA synthetase
Ibba M, Soll D. (2000) Aminoacyl-tRNA synthesis. Annu Rev Biochem. 69, 617-650.
Ibba M, Stathopoulos C, Soll D. (2001) Protein synthesis: twenty three amino acids and counting. Curr Biol. 11, R563-565.
Ibba M, Soll D. (2001) The renaissance of aminoacyl-tRNA synthesis. EMBO Rep. 5, 382-387.
Sekine S, Nureki O, Dubois DY, Bernier S, Chenevert R, Lapointe J, Vassylyev DG, Yokoyama S. (2003) ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding. EMBO J. 22, 676-88.
Schulze JO, Masoumi A, Nickel D, Jahn M, Jahn D, Schubert WD, Heinz DW. (2006) Crystal Structure of a Non-discriminating Glutamyl-tRNA Synthetase. J Mol Biol. 361, 888-897.
Date/time: Wednesday, 25 October, 15:30
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