Abstract
Uridine phosphorylase (UP) is a key enzyme of pyrimidine salvage pathways that enables the recycling of endogenous or exogenous-supplied pyrimidines and plays an important intracellular metabolic role. Here, we biochemically and structurally characterized two evolutionarily divergent uridine phosphorylases, PcUP1 and PcUP2 from the oomycete pathogen Phytophthora capsici. Our analysis of other oomycete genomes revealed that both uridine phosphorylases are present in Phytophthora and Pythium genomes, but only UP2 is seen in Saprolegnia spp. which are basal members of the oomycetes. Moreover, uridine phosphorylases are not found in obligate oomycete pathogens such as Hyaloperonospora arabidopsidis and Albugo spp. PcUP1 and PcUP2 are upregulated 300 and 500 fold respectively, within 90 min after infection of pepper leaves. The crystal structures of PcUP1 in ligand-free and in complex with uracil/ribose-1-phosphate, 2′-deoxyuridine/phosphate and thymidine/phosphate were analyzed. Crystal structure of this uridine phosphorylase showed strict conservation of key residues in the binding pocket. Structure analysis of PcUP1 with bound ligands, and site-directed mutagenesis of key residues provide additional support for the “push-pull” model of catalysis. Our study highlights the importance of pyrimidine salvage during the earliest stages of infection.
Highlights
Uridine phosphorylase (UP) is a key enzyme of pyrimidine salvage pathways that enables the recycling of endogenous or exogenous-supplied pyrimidines and plays an important intracellular metabolic role
The high level of upregulation of PcUP1 and PcUP2 within 90 min after infection suggests that oomycete hyphae are already utilizing nucleotides that are exported from host tissues, a strategy that is not available for obligate oomycete pathogens, since these enzymes are not present in those genomes
The expression patterns for oomycete enzymes in the pyrimidine biosynthesis and salvage pathways were first described for the potato pathogen P. infestans[7]
Summary
Uridine phosphorylase (UP) is a key enzyme of pyrimidine salvage pathways that enables the recycling of endogenous or exogenous-supplied pyrimidines and plays an important intracellular metabolic role. The crystal structures of PcUP1 in ligand-free and in complex with uracil/ribose-1-phosphate, 2′-deoxyuridine/phosphate and thymidine/ phosphate were analyzed Crystal structure of this uridine phosphorylase showed strict conservation of key residues in the binding pocket. While the enzymatic strategies for synthesis of purine and pyrimidine are strongly conserved across the domains of life, different salvage pathways are seen in mammals, plants, and fungi[5,6]. Uridine phosphorylase (UP) (E.C. 2.4.2.3), a key enzyme involved in the pyrimidine salvage pathway, in mammals and some bacteria, catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate[8]. Our structural analysis of key conserved residues surrounding the enzymatic pocket provides additional insight on the catalytic mechanism of UPs. This study describes two examples of widely divergent UPs in Pythium and Phytophthora genomes to optimally utilize nucleotide resources under different conditions. Transcriptomic analysis of very early time points following infection of leaves with zoospores may lead to the identification of effectors mediating this process
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