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Plants can catabolize purine nucleotides to recycle nutrients, in particular nitrogen. The currently established model of purine nucleotide catabolism consists of a branched pathway that starts from AMP and GMP and proceeds either via the intermediates inosine and hypoxanthine or via guanosine and xanthosine to converge on xanthine. Xanthine is further catabolized in a linear fashion by a fully characterized series of reactions to form glyoxylate, carbon dioxide and ammonia. The ammonia released can be re-assimilated into amino acids. This work focuses on how xanthine is generated, in vivo, in Arabidopsis. Metabolite analysis of mutants of a wide set of genes involved in purine catabolism and salvage such as guanosine deaminase, nucleoside hydrolases (NSH), xanthine dehydrogenase, urate oxidase and hypoxanthine guanine phosphoribosyltransferase along with double order and triple order mutants of the same genes showed that xanthine is mainly generated by xanthosine hydrolysis. Inosine and hypoxanthine might enter the pathway from routes other than nucleotide degradation, such as tRNA degradation, DNA repair, and uptake from the soil. Furthermore, xanthosine is not only generated by guanosine deamination as reported in Dahncke and Witte, 2013, but xanthosine monophosphate dephosphorylation is likely to be a source as well. In addition, this work elucidates the function of NSH2. The Arabidopsis genome encodes two nucleoside hydrolases, NSH1 and NSH2. NSH1 is essential for xanthosine and uridine hydrolysis, whereas the function of NSH2 is unclear. Biochemical, genetic and metabolic analyses demonstrate that NSH1 activates NSH2 in vitro and in vivo forming a heterocomplex that has a higher catalytic efficiency for xanthosine, but not for uridine, in comparison to the NSH1 homomer. The heterocomplex formation is also shown for the NSH enzymes of Coffea arabica and Physcomitrella patens, suggesting that this interaction is conserved in the plant kingdom. Dynamic NSH heterocomplex formation might regulate the flux ...