Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean

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

Research output: Contribution to journalArticle (Academic Journal)peer-review

68 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)418-30
Number of pages13
JournalPlant Physiology
Volume146
Issue number2
DOIs
Publication statusPublished - Feb 2008

Keywords

  • Amidohydrolases
  • Arabidopsis
  • Escherichia coli
  • Gene Expression Regulation, Plant
  • Genetic Complementation Test
  • Kinetics
  • Metals
  • Molecular Sequence Data
  • Mutation
  • Plant Proteins
  • Protein Transport
  • Soybeans
  • Ureohydrolases
  • Journal Article
  • Research Support, Non-U.S. Gov't

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