Characterisation of the first wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase resembling mammalian counterparts |
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Authors: | Iris J Joye Tim Beliën Kristof Brijs Paul Proost Wim Soetaert Jan A Delcour |
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Institution: | 1. Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium;2. Laboratory of Molecular Immunology, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium;3. Laboratory of Industrial Microbiology and Biocatalysis, Universiteit Gent, Coupure links 653, B-9000 Ghent, Belgium |
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Abstract: | A formerly unknown nucleotide pyrophosphatase/phosphodiesterase (NPP) of wheat (Triticum aestivum L.) (TaNPP) was recombinantly produced in Pichia pastoris (TaNPPr) following assembly of the sequence out of wheat ESTs using sequences of known NPPs. Simultaneously, a phosphodiesterase was purified to homogeneity from wheat germs, characterised and identified as TaNPP. TaNPP contains the highly conserved catalytic substrate and metal binding residues and displays properties high pH optimum, glycosylation, high thermostability and inhibition by EDTA and adenosine 5′-monophosphate (AMP)] similar to those of mammalian enzymes characterised earlier. The sequence of TaNPP includes that of a putative transmembrane domain, a characteristic of most NPPs. Both recombinant and native TaNPP partially occur as oligomeric proteins on SDS PAGE. Upon addition of DTT, both migrate as monomeric proteins. Part of the native wheat protein even occurs in its truncated form, thus lacking the transmembrane region. Out of a range of tested natural substrates, TaNPP had the highest affinity towards adenine containing nucleotides. While Michaelis-Menten kinetics were valid in the low substrate concentration range, at higher concentrations, they were no longer applicable. TaNPP shows no similarity to the recently characterised rice and barley enzymes and, is hence, one of the first characterised plant NPPs resembling mammalian NPPs. |
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Keywords: | Nucleotide pyrophosphatase/phosphodiesterase Wheat Nicotinamide coenzymes |
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