title: Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism.
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.
authors.affiliation: Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001, Leuven, Belgium. iris.joye@biw.kuleuven.be
authors.forename: Iris J
authors.initials: IJ
authors.lastname: Joye
authors.forename: Tim
authors.initials: T
authors.lastname: Beliën
authors.forename: Kristof
authors.initials: K
authors.lastname: Brijs
authors.forename: Wim
authors.initials: W
authors.lastname: Soetaert
authors.forename: Jan A
authors.initials: JA
authors.lastname: Delcour
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014908:Triticum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase