Biochemical characterization, homology modeling and docking studies of ornithine delta-aminotransferase--an important enzyme in proline biosynthesis of plants.
Ornithine delta-aminotransferase (OAT) is an important enzyme in proline biosynthetic pathway and is implicated in salt tolerance in higher plants. OAT transaminates ornithine to pyrroline 5-carboxylate, which is further catalyzed to proline by pyrroline 5-carboxylate reductase. The Vigna aconitifolia OAT cDNA, encoding a polypeptide of 48.1 kDa, was expressed in Escherichia coli and the enzyme was partially characterized following its purification using (NH(4))(2)SO(4) precipitation and gel filtration techniques. Optimal activity of the enzyme was observed at a temperature of 25 degrees C and pH 8.0. The enzyme appeared to be a monomer and exhibited high activity at 4mM ornithine. Proline did not show any apparent effect but isoleucine, valine and serine inhibited the activity when added into the assay mixture along with ornithine. Omission of pyridoxal 5'-phosphate from the reaction mixture reduced the activity of this enzyme by 60%. To further evaluate these biochemical observations, homology modeling of the OAT was performed based on the crystal structure of the ornithine delta-aminotransferase from humans (PDB code 1OAT) by using the software MODELLER6v2. With the aid of the molecular mechanics and dynamics methods, the final model was obtained and assessed subsequently by PROCHECK and VERIFY-3D graph. With this model, a flexible docking study with the substrate and inhibitors was performed and the results indicated that Gly106 and Lys256 in OAT are the important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate and inhibitors. These observations are in conformity with the results obtained from experimental investigations.
Sekhar
P Nataraj
PN
Department of Genetics, Osmania University, Hyderabad 500007, India.
Amrutha
R Naga
RN
Sangam
Shubhada
S
Verma
D P S
DP
Kishor
P B Kavi
PB
D000595:Amino Acid Sequence; D003198:Computer Simulation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009953:Ornithine-Oxo-Acid Transaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000201:enzymology / Q000378:metabolism*; D011392:Proline / Q000096:biosynthesis*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D011392:Proline; D009953:Ornithine-Oxo-Acid Transaminase