Identification, Biochemical Characterization, and Subcellular Localization of Allantoate Amidohydrolases from Arabidopsis and Soybean

Abstract

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 mM 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.

Identification of new QTLs for seed mineral, cysteine, and methionine concentrations in soybean [Glycine max (L.) Merr.]

“…An allantoate amidohydrolase gene (AAH) was detected in the N QTL interval on chromosome 15. AAHs are localized in the endoplasmic reticulum, and they hydrolyze the ureide allantoate to produce ureidoglycolate, CO 2 and two molecules of ammonium (Werner et al 2008). Allantoate degradation is required for recycling of purine-ring N in all plants.…”

“…Allantoate degradation is required for recycling of purine-ring N in all plants. In some tropical legumes such as soybean, fixed N is transported through allantoin and then through allantoate in shoots, where it serves as a general N source (Werner et al 2008). On chromosome 20, homocysteine S-methyltransferase (HMT3) was present in the QTL interval for Cys + Met.…”

Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis

“…Nicotiana benthamiana was grown and transgenic A. thaliana generated as described by Witte et al (2004). Transient expression in N. benthamiana was performed as described by Werner et al (2008). For confocal microscopy, YFP-AtGSDA and b-UP-CFP (for b-ureidopropionase-cyan fluorescent protein) were coexpressed by mixing the respective transgenic Agrobacterium tumefaciens strains and agrobacteria expressing the p19 silencing inhibitor from tomato bushy stunt virus.…”

Ureide Content of Guar under Influence of Hexaconazole and Triazophos

“…The occurrence of several pathways for the degradation of both allantoate and ureidoglycolate has been reported [16,17]. Most recent reports suggested that plants degrade allantoate to ureidoglycolate via allantoate amidohydrolase (AAH; EC 3.5.3.9) and ureidoglycine aminohydrolase (EC 3.5.3-) [18][19][20][21][22]. The ureidic plants relying upon N 2 fixation as the sole nitrogen source, ureides may comprise up to 86% of the N in the xylem sap, whereas amino acids, amides and nitrate are the major forms of nitrogen translocated from the roots after fertilization with nitrate [23,24].…”