Molecular and functional characterization of allantoate amidohydrolase from Phaseolus vulgaris.
Allantoate degradation is an essential step for recycling purine-ring nitrogen in all plants, but especially in tropical legumes where the ureides allantoate and allantoin are the main compounds used to store and transport the nitrogen fixed in nodules. Two enzymes, allantoate amidohydrolase (AAH) and allantoate amidinohydrolase (allantoicase), could catalyze allantoate breakdown, although only AAH-coding sequences have been found in plant genomes, whereas allantoicase-related sequences are restricted to animals and some microorganisms. A cDNA for AAH was cloned from Phaseolus vulgaris leaves. PvAAH is a single-copy gene encoding a polypeptide of 483 amino acids that conserves all putative AAH active-site domains. Expression and purification of the cDNA in Nicotiana benthamiana showed that the cloned sequence is a true AAH protein that yields ureidoglycine and ammonia, with a Km of 0.46 mM for allantoate. Optimized in vitro assay, quantitative RT-PCR and antibodies raised to the PvAAH protein were used to study AAH under physiological conditions. PvAAH is ubiquitously expressed in common bean tissues, although the highest transcript levels were found in leaves. In accordance with the mRNA expression levels, the highest PvAAH activity and allantoate concentration also occurred in the leaves. Comparison of transcript levels, protein amounts and enzymatic activity in plants grown with different nitrogen sources and upon drought stress conditions showed that PvAAH is regulated at posttranscriptional level. Moreover, RNAi silencing of AAH expression increases allantoate levels in the transgenic hairy roots, indicating that AAH should be the main enzyme involved in allantoate degradation in common bean.
Departamento de Botánica, Ecología y Fisiología Vegetal, Campus de Excelencia Internacional Agroalimentario (CEIA3), Campus de Rabanales, Edif. Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071x0, Córdoba, Spain.
Juan Luis
JL
Díaz-Leal
Fernando
F
Torralbo
Francisco
F
Antonio Quiles
Manuel
M
Pineda
Josefa M
JM
Alamillo
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D055864:Droughts; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism*; D009928:Organ Specificity; D027805:Phaseolus / Q000201:enzymology* / Q000235:genetics; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D030821:Plants, Genetically Modified; D018749:RNA, Plant / Q000235:genetics; D011817:Rabbits; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000031:analogs & derivatives / Q000378:metabolism; D014512:Ureohydrolases / Q000235:genetics* / Q000378:metabolism
D000906:Antibodies; D018076:DNA, Complementary; D018749:RNA, Plant; C024081:allantoic acid; D014508:Urea; D014512:Ureohydrolases; C019657:allantoate amidohydrolase; D009584:Nitrogen