In Silico Characterization and Homology Modeling of Thylakoid bound Ascorbate Peroxidase from a Drought Tolerant Wheat Cultivar

Ascorbate peroxidase, a haem protein (EC 1.11.1.11), efficiently scavenges hydrogen peroxide (H2O2) in cytosol and chloroplasts of plants. In this study, a full-length coding sequence of thylakoid-bound ascorbate peroxidase cDNA (TatAPX) was cloned from a drought tolerant wheat cultivar C306. Homology modeling of the TatAPX protein was performed by using the template crystal structure of chloroplastic ascorbate peroxidase from tobacco plant (PDB: 1IYN). The model structure was further refined by molecular mechanics and dynamic methods using various tools such as PROCHECK, ProSA and Verify3D. The predicted model was then tested for docking with H2O2, the substrate for TatAPX enzyme. The results revealed that Arg233 and Glu255 in the predicted active site of the enzyme are two important amino acid residues responsible for strong hydrogen bonding affinity with H2O2, which might play an important role in scavenging of H2O2 from the plant system.

[1]  D. Eisenberg,et al.  Assessment of protein models with three-dimensional profiles , 1992, Nature.

[2]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[3]  H. Hirt,et al.  Reactive oxygen species: metabolism, oxidative stress, and signal transduction. , 2004, Annual review of plant biology.

[4]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[5]  Song Liu,et al.  Protein binding site prediction using an empirical scoring function , 2006, Nucleic acids research.

[6]  D. van der Spoel,et al.  GROMACS: A message-passing parallel molecular dynamics implementation , 1995 .

[7]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[8]  S. Sinha,et al.  Assessment of drought resistance in 20 accessions of Triticum aestivum L. and related species II. Stability in yield components , 1991, Euphytica.

[9]  Ben M. Webb,et al.  Comparative Protein Structure Modeling Using Modeller , 2006, Current protocols in bioinformatics.

[10]  G J Kleywegt,et al.  Phi/psi-chology: Ramachandran revisited. , 1996, Structure.

[11]  Manfred J. Sippl,et al.  Thirty years of environmental health research--and growing. , 1996, Nucleic Acids Res..

[12]  Takeshi Kawabata,et al.  MATRAS: a program for protein 3D structure comparison , 2003, Nucleic Acids Res..

[13]  Robert D. Clark,et al.  SYBYL Line Notation (SLN): A Single Notation To Represent Chemical Structures, Queries, Reactions, and Virtual Libraries , 2008, J. Chem. Inf. Model..

[14]  J. Thornton,et al.  Stereochemical quality of protein structure coordinates , 1992, Proteins.

[15]  Mark Johnson,et al.  NCBI BLAST: a better web interface , 2008, Nucleic Acids Res..

[16]  R. Ugalde,et al.  Thylakoid-Bound Ascorbate Peroxidase Mutant Exhibits Impaired Electron Transport and Photosynthetic Activity1 , 2003, Plant Physiology.

[17]  J. P. Jenuth.  The NCBI. Publicly available tools and resources on the Web. , 2000, Methods in molecular biology.

[18]  H. Edelsbrunner,et al.  Anatomy of protein pockets and cavities: Measurement of binding site geometry and implications for ligand design , 1998, Protein science : a publication of the Protein Society.

[19]  S. Sinha,et al.  Assessment of drought resistance in 20 accessions of Triticum aestivum and related species I. Total dry matter and grain yield stability , 1991, Euphytica.

[20]  N. Suzuki,et al.  Double Mutants Deficient in Cytosolic and Thylakoid Ascorbate Peroxidase Reveal a Complex Mode of Interaction between Reactive Oxygen Species, Plant Development, and Response to Abiotic Stresses1[W][OA] , 2007, Plant Physiology.

[21]  Julie D Thompson,et al.  Multiple Sequence Alignment Using ClustalW and ClustalX , 2003, Current protocols in bioinformatics.

[22]  C. Kao,et al.  Expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativa L.) seedlings in response to NaCl. , 2007, Journal of experimental botany.

[23]  S. Shigeoka,et al.  cDNAs encoding spinach stromal and thylakoid‐bound ascorbate peroxidase, differing in the presence or absence of their 3′‐coding regions , 1996, FEBS letters.

[24]  Piero Carninci,et al.  Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stresses by Using a Full-Length cDNA Microarray , 2001, Plant Cell.

[25]  K. Asada.  Production and Scavenging of Reactive Oxygen Species in Chloroplasts and Their Functions1 , 2006, Plant Physiology.

[26]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[27]  Richard M. Jackson,et al.  Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites , 2005, Bioinform..

[28]  K. Asada.  Ascorbate peroxidase – a hydrogen peroxide‐scavenging enzyme in plants , 1992 .

[29]  Prosenjit Mondal,et al.  In Silico Modeling and Hydrogen Peroxide Binding Study of Rice Catalase , 2006, Silico Biol..

[30]  D. Eisenberg,et al.  VERIFY3D: assessment of protein models with three-dimensional profiles. , 1997, Methods in enzymology.

[31]  Ricardo L. Mancera,et al.  Molecular modelling prediction of ligand binding site flexibility , 2004, J. Comput. Aided Mol. Des..

[32]  Aleksey A. Porollo,et al.  Enhanced recognition of protein transmembrane domains with prediction-based structural profiles , 2006, Bioinform..

[33]  A. Sali,et al.  Comparative protein structure modeling of genes and genomes. , 2000, Annual review of biophysics and biomolecular structure.

[34]  Y. Yao,et al.  The 3D structure of the defense-related rice protein Pir7b predicted by homology modeling and ligand binding studies , 2008, Journal of molecular modeling.

[35]  Stavros J. Hamodrakas,et al.  waveTM: Wavelet-based transmembrane segment prediction , 2004, Silico Biol..

[36]  István Simon,et al.  The HMMTOP transmembrane topology prediction server , 2001, Bioinform..

[37]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[38]  M. Hayashi,et al.  cDNA cloning of thylakoid-bound ascorbate peroxidase in pumpkin and its characterization. , 1996, Plant & cell physiology.

[39]  A. Sali,et al.  Modeling of loops in protein structures , 2000, Protein science : a publication of the Protein Society.

[40]  M. Tamoi,et al.  Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach. , 1998, Plant & cell physiology.