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Published on January 28, 2009; 10.1104/pp.108.134510

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Received December 17, 2008
Accepted January 23, 2009

Molecular Modeling and Site-Directed Mutagenesis Reveal Essential Residues for Catalysis in a Prokaryotic-Type Aspartate Aminotransferase

Fernando de la Torre , Aurelio A. Moya-Garcia , Maria-Fernanda Suarez , Carlos Rodriguez-Caso , Rafael A. Canas , Francisca Sanchez-Jimenez , and Francisco M. Canovas *

Departamento de Biologia Molecular y Bioquimica and Instituto Andaluz de Biotecnologia, Departamento de Biologia Molecular y Bioquimica and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Campus Universitario de Teatinos, Universidad de Malaga, 29071-Malaga, Spain; and ICREA-Complex Systems Lab. Barcelona Biomedical Research Park. Dr. Aiguader 88, 08003 Barcelona, Spain

* Corresponding author; email: canovas{at}uma.es.

We recently reported that aspartate biosynthesis in plant chloroplasts is catalyzed by two different aspartate aminotransferases (AAT, EC 2.6.1.1): a previously characterized eukaryotic-type and a prokaryotic-type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryotic-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 aspartate-derived amino acids inside the organelle. In the present 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, PDB 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 Thermus termophilus such as those involved in the recognition of the substrate side-chain or the "open to close" transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses and several critical residues (V206, S207, Q346, E210 and F450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.




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