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Title: Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism
Authors: Joye, Iris ×
Beliën, Tim
Brijs, Kristof
Soetaert, Wim
Delcour, Jan #
Issue Date: 2011
Publisher: Springer-Verlag
Series Title: Applied Microbiology and Biotechnology vol:90 pages:173-180
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.
ISSN: 0175-7598
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Centre for Food and Microbial Technology
× corresponding author
# (joint) last author

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