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Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism

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Abstract
Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5'-monophosphates from a wide range of nucleotide substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51: 326-336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326-336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates. Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5'-monophosphate-containing substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very stringent.
Keywords
Nucleotide pyrophosphatase/phosphodiesterase, Alkaline phosphatase, Mutagenesis, FAMILY, PYROPHOSPHATASES/PHOSPHODIESTERASES, SUPERFAMILY

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MLA
Joye, Iris J, Tim Belien, Kristof Brijs, et al. “Mutational Analysis of Wheat (Triticum Aestivum L.) Nucleotide Pyrophosphatase/phosphodiesterase Shows the Role of Six Amino Acids in the Catalytic Mechanism.” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 90.1 (2011): 173–180. Print.
APA
Joye, I. J., Belien, T., Brijs, K., Soetaert, W., & Delcour, J. A. (2011). Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 90(1), 173–180.
Chicago author-date
Joye, Iris J, Tim Belien, Kristof Brijs, Wim Soetaert, and Jan A Delcour. 2011. “Mutational Analysis of Wheat (Triticum Aestivum L.) Nucleotide Pyrophosphatase/phosphodiesterase Shows the Role of Six Amino Acids in the Catalytic Mechanism.” Applied Microbiology and Biotechnology 90 (1): 173–180.
Chicago author-date (all authors)
Joye, Iris J, Tim Belien, Kristof Brijs, Wim Soetaert, and Jan A Delcour. 2011. “Mutational Analysis of Wheat (Triticum Aestivum L.) Nucleotide Pyrophosphatase/phosphodiesterase Shows the Role of Six Amino Acids in the Catalytic Mechanism.” Applied Microbiology and Biotechnology 90 (1): 173–180.
Vancouver
1.
Joye IJ, Belien T, Brijs K, Soetaert W, Delcour JA. Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2011;90(1):173–80.
IEEE
[1]
I. J. Joye, T. Belien, K. Brijs, W. Soetaert, and J. A. Delcour, “Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism,” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 90, no. 1, pp. 173–180, 2011.
@article{1220946,
  abstract     = {Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5'-monophosphates from a wide range of nucleotide substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51: 326-336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326-336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates. Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5'-monophosphate-containing substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very stringent.},
  author       = {Joye, Iris J and Belien, Tim and Brijs, Kristof and Soetaert, Wim and Delcour, Jan A},
  issn         = {0175-7598},
  journal      = {APPLIED MICROBIOLOGY AND BIOTECHNOLOGY},
  keywords     = {Nucleotide pyrophosphatase/phosphodiesterase,Alkaline phosphatase,Mutagenesis,FAMILY,PYROPHOSPHATASES/PHOSPHODIESTERASES,SUPERFAMILY},
  language     = {eng},
  number       = {1},
  pages        = {173--180},
  title        = {Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism},
  url          = {http://dx.doi.org/10.1007/s00253-010-2962-z},
  volume       = {90},
  year         = {2011},
}

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