Structural and catalytic analysis of two diverse uridine phosphorylases in Phytophthora capsici

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. Phytophthora capsici, one of the most destructive plant pathogens of Phytophthora spp., causes economic losses to tomatoes, peppers, cucurbits, lima, and snap bean crops 1. The most effective control strategies of Phytophthora require a combination of different methods 1. However, chemical control of Phytophthora, is becoming less effective because of growing resistance to existing chemicals, and because oomycetes lack the targets for most of the broad-spectrum agrochemicals deployed against phytopathogenic fungi. Since metabolism and nutrient acquisition play an important role in disease establishment, infection, and development of plant pathogens 2 a complete, detailed analysis of metabolic strategies used by foliar oomycete pathogens is warranted to identify new strategies for control of these pathogens 3. The biosynthesis of purine and pyrimidine nucleotides which are critical substrates for growth and replication, can proceed via de novo synthesis from amino acids, or by salvage pathways that enable the recycling of bases and nucleosides formed during the degradation of RNA and DNA 4. 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 Earlier work indicated that during the initial biotrophic phase of the foliar infection by the potato blight pathogen P. infestans, biosynthesis of pyrimidines was strongly upregulated, indicating that invasive growth was not dependent on plant nucleotides, while the salvage pathway appeared to become more important during the necrotrophic stage of infection 7. The nucleotide salvage pathway has also b...

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“…In bacteria and mammals an enzyme termed uridine phosphorylase (EC: 2.4.2.3) catalyzes the reversible conversion of uridine into uracil [ 24 , 25 ]. Only recently, two proteins with this enzymatic activity, PcUP1 and PcUP2, have been characterized in the plant pathogenic Oomycete Phytophthora capsici [ 26 ]. In the next step we looked for putative uridine phosphorylase orthologs using the corresponding Escherichia coli protein sequence Udp [ 27 ], mammalian UPP1 and UPP2 [ 28 , 29 ] and that of PcUP1 and PcUP2.…”

“…In bacteria and mammals an enzyme termed uridine phosphorylase (EC: 2.4.2.3) catalyzes the reversible conversion of uridine into uracil [24,25]. Only recently, two proteins with this enzymatic activity, PcUP1 and PcUP2, have been characterized in the plant pathogenic Oomycete Phytophthora capsici [26].…”

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