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Molecular modeling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase.

de la Torre, F; Moya-García, AA; Suárez, M-F; Rodríguez-Caso, C; Cañas, RA; Sánchez-Jiménez, F; Cánovas, FM; (2009) Molecular modeling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase. Plant Physiol , 149 (4) pp. 1648-1660. 10.1104/pp.108.134510.

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Abstract

We recently reported that aspartate (Asp) biosynthesis in plant chloroplasts is catalyzed by two different Asp aminotransferases (AAT): a previously characterized eukaryote type and a prokaryote type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryote-type AAT is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the PT-AAT could be involved in the biosynthesis of the Asp-derived amino acids inside the organelle. In this work, a comparative modeling of the PT-AAT enzyme from Pinus pinaster (PpAAT) was performed using x-ray structures of a bacterial AAT (Thermus thermophilus; Protein Data Bank accession nos. 1BJW and 1BKG) as templates. We computed a three-dimensional folding model of this plant homodimeric enzyme that has been used to investigate the functional importance of key amino acid residues in its active center. The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. Moreover, PpAAT monomers folded into one large and one small domain. However, PpAAT enzyme showed unique structural and functional characteristics that have been specifically described in the AATs from the prokaryotes Phormidium lapideum and T. thermophilus, such as those involved in the recognition of the substrate side chain or the "open-to-closed" transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.

Type: Article
Title: Molecular modeling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase.
Location: United States
DOI: 10.1104/pp.108.134510
Keywords: Amino Acid Sequence, Amino Acids, Aspartate Aminotransferases, Biocatalysis, Catalytic Domain, Kinetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutant Proteins, Pinus, Prokaryotic Cells, Protein Structure, Secondary, Protein Structure, Tertiary, Pyridoxal Phosphate, Recombinant Proteins, Sequence Alignment, Spectrophotometry, Substrate Specificity, Thermus thermophilus
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1432049
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