Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean.
Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO(2), 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 a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 microm manganese are consistent with allantoate being the in vivo substrate of manganese-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 nitrogen 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 localize to the endoplasmic reticulum 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 endoplasmic reticulum.
Freie Universität Berlin, Institut für Biologie, Abteilung Biochemie der Pflanzen, 14195 Berlin, Germany.
Andrea K
AK
Werner
Imogen A
IA
Sparkes
Tina
T
Romeis
Claus-Peter
CP
Witte
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D007700:Kinetics; D008670:Metals; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D013025:Glycine max / Q000201:enzymology*; D014512:Ureohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D008670:Metals; D010940:Plant Proteins; D000581:Amidohydrolases; C019659:allantoinase; D014512:Ureohydrolases; C019657:allantoate amidohydrolase