Abstract
A central step in nucleoside and nucleobase salvage pathways is the hydrolysis of nucleosides to their respective nucleobases. In plants this is solely accomplished by nucleosidases (EC 3.2.2.x).To elucidate the importance of nucleosidases for nucleoside degradation, general metabolism, and plant growth, thorough phenotypic and biochemical analyses were performed using Arabidopsis thaliana T-DNA insertion mutants lacking expression of the previously identified genes annotated as uridine ribohydrolases (URH1 and URH2).Comprehensive functional analyses of single and double mutants demonstrated that both isoforms are unimportant for seedling establishment and plant growth, while one participates in uridine degradation. Rather unexpectedly, nucleoside and nucleotide profiling and nucleosidase activity screening of soluble crude extracts revealed a deficiency of xanthosine and inosine hydrolysis in the single mutants, with substantial accumulation of xanthosine in one of them. Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, thus providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis.This mutant study demonstrates the utility of in vivo systems for the examination of metabolic activities, with the discovery of the new substrate xanthosine and elucidation of a mechanism for expanding the nucleosidase substrate spectrum.
Highlights
Nucleotides and nucleic acids were among the first spontaneously synthesized biomolecules (Powner et al, 2009), and they play an important role in evolution, information preserving and catalytic activity (Joyce, 2002)
Mixing of the two mutant extracts, and by in vitro activity reconstitution using a mixture of recombinant URH1 and URH2 proteins, both restored activity, providing biochemical evidence that at least these two isoforms are needed for inosine and xanthosine hydrolysis
Ribonucleoside hydrolase activity was quantified in crude extracts of roots and leaves by determination of uracil, hypoxanthine or xanthine originating from added uridine, inosine or xanthosine, respectively
Summary
Nucleotides and nucleic acids were among the first spontaneously synthesized biomolecules (Powner et al, 2009), and they play an important role in evolution, information preserving and catalytic activity (Joyce, 2002). As an evolutionarily ancient and indispensable complex of metabolic pathways, the metabolism of both classes of nucleotides in plants is comparable to that in other organisms (Moffatt & Ashihara, 2002; Zrenner et al, 2006). It starts with the formation of central metabolites, while all purines are derived from IMP (van der Graaff et al, 2004) and all pyrimidines from UMP (Giermann et al, 2002; Schroder et al, 2005). The whole range of nucleotides is generated by amination, desamination and oxidation of the heterocyclic ring system
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