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dc.contributor.authorAgren, Daniel
dc.contributor.authorStehr, Matthias
dc.contributor.authorBerthold, Catrine L
dc.contributor.authorKapoor, Shobhna
dc.contributor.authorOehlmann, Wulf
dc.contributor.authorSingh, Mahavir
dc.contributor.authorSchneider, Gunter
dc.date.accessioned2009-06-22T13:53:17Z
dc.date.available2009-06-22T13:53:17Z
dc.date.issued2008-04-04
dc.identifier.citationThree-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding. 2008, 377 (4):1161-73 J. Mol. Biol.en
dc.identifier.issn1089-8638
dc.identifier.pmid18304579
dc.identifier.doi10.1016/j.jmb.2008.01.091
dc.identifier.urihttp://hdl.handle.net/10033/71163
dc.description.abstractL-alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to L-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 A resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16 degrees toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.
dc.language.isoenen
dc.subject.meshAlanine Dehydrogenaseen
dc.subject.meshApoenzymesen
dc.subject.meshCatalysisen
dc.subject.meshCoenzymesen
dc.subject.meshEnzyme Activationen
dc.subject.meshHoloenzymesen
dc.subject.meshImaging, Three-Dimensionalen
dc.subject.meshModels, Biologicalen
dc.subject.meshModels, Molecularen
dc.subject.meshMutagenesis, Site-Directeden
dc.subject.meshMycobacterium tuberculosisen
dc.subject.meshNADen
dc.subject.meshProtein Bindingen
dc.subject.meshProtein Structure, Quaternaryen
dc.subject.meshProtein Subunitsen
dc.subject.meshPyruvic Aciden
dc.titleThree-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding.en
dc.typeArticleen
dc.contributor.departmentDepartment of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.en
dc.identifier.journalJournal of molecular biologyen
refterms.dateFOA2018-06-12T23:12:40Z
html.description.abstractL-alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to L-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 A resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16 degrees toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.


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