Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation

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.

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

“…Isolated NSH2 has not yet been characterized biochemically, but extracts of an nsh2 mutant line show strongly reduced xanthosine hydrolase activity (Riegler et al, 2011). However, the nsh2 mutant does not accumulate xanthosine as observed in the nsh1 mutant (Jung et al, 2011;Riegler et al, 2011).…”

“…It was recently reported that mutation of NSH1 (formerly URH1) of A. thaliana leads to accumulation of xanthosine and uridine in this plant because the enzyme is required for the hydrolysis of both compounds in vivo (Jung et al, 2011;Riegler et al, 2011). We crossed plants carrying the published nsh1-1 allele (SALK_083120) with plants possessing the gsda-2 allele to assess the contribution of GSDA to the xanthosine pool in vivo.…”

“…The Rib moiety of guanosine (and other nucleosides) is removed either by phosphorolysis or hydrolysis, giving rise to (1) guanine (and other nucleobases) and to (2) Rib 1-phosphate or Rib, respectively. Arabidopsis thaliana lacks nucleoside phosphorylases but possesses two intracellular nucleoside hydrolases (NUCLEOSIDE HYDROLASE1 [NSH1] and NSH2) with overlapping function in purine and pyrimidine nucleoside catabolism (Jung et al, 2009(Jung et al, , 2011Riegler et al, 2011). However, it has not been shown that these enzymes hydrolyze guanosine.…”

Glutaredoxin GRXS17 Associates with the Cytosolic Iron-Sulfur Cluster Assembly Pathway

“…URH1 and URH2 are nucleoside ribohydrolases with inosine and xanthosine hydrolytic activity, leading to hypoxanthine and xanthine generation (Riegler et al, 2011). Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, which are the final products of purine degradation.…”

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

“…However, it is unclear whether or how uridine is imported into plastids. Pyrimidine and purine-nucleoside cleavage is mainly facilitated by nucleoside hydrolase1 (NSH1), a cytosolic enzyme (Jung et al, 2009, Riegler et al, 2011). The resulting nucleobases can then undergo either salvage by phosphoribosyl-pyrophosphatedependent phosphoribosyl-transferases or catabolism.…”