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Published on December 7, 2007; 10.1104/pp.107.110809

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Received October 19, 2007
Accepted November 26, 2007

Identification, biochemical characterisation, and subcellular localisation of allantoate amidohydrolases from Arabidopsis thaliana and Glycine max

Andrea K. Werner , Imogen A. Sparkes , Tina Romeis , and Claus-Peter Witte *

Freie Universitat Berlin, Institut fur Biologie, Abteilung Biochemie der Pflanzen, Konigin-Luise-Str. 12-16, 14195 Berlin, Germany; Oxford Brookes University, School of Life Sciences, Gipsy Lane, Oxford OX3 0BP, United Kingdom

* Corresponding author; email: cpwitte{at}zedat.fu-berlin.de.

Allantoate amidohydrolases hydrolise the ureide allantoate to ureidoglycolate, carbon dioxide and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot where it serves as general N source.

Allantoate amidohydrolases from Arabidopsis thaliana (AtAAH) and from Glycine max (GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release two mol of ammonium per mol allantoate. Therefore they can truly be classified as allantoate amidohydrolases. The kinetic constants determined and the half-maximal activation by 2 to 3 µM manganese are consistent with allantoate being the in-vivo substrate of Mn-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate and by millimolar concentrations of L-asparagine and L-aspartate but not D-asparagine. L-asparagine likely functions as competitive inhibitor.

An Ataah T-DNA mutant, unable to grow on allantoin as sole N source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localise to the endoplasmic reticulum (ER) after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the ER.






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