Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs

Soysa, Radika; Wilson, Zachary N; Elferich, Johannes; Forquer, Isaac; Shinde, Ujwal; Riscoe, Michael K.; Yates, Phillip A.; Ullman, Buddy

doi:10.1074/jbc.m113.478826

Cited by 12 publications

(13 citation statements)

References 31 publications

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“…Characterization of recombinant AgUPRT revealed a type of metabolic enzyme regulation previously unknown in A. gossypii. Moreover, common features with other organisms, such as unrelated protozoan parasites [13,14] and other flavinogenic fungi, were identified and discussed, thus extending the impact of this study.…”

Section: Introductionsupporting

confidence: 52%

“…AgUPRT enzyme activity was determined spectrophotometrically by measuring the conversion of uracil into UMP as previously described [14,20]. To determine the effect of uracil concentration on AgUPRT activity, the assay mixture consisted of TMD 50 buffer (50 mM Tris−HCl, 5 mM MgCl 2 , and 2 mM DTT, pH 7.5) with 1 mM phosphoribosyl pyrophosphate (PRPP) and uracil concentrations ranging from 10 μM to 1.25 mM.…”

Section: Enzyme Activity Assaysmentioning

confidence: 99%

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Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii

et al. 2019

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The blockage of the de novo pyrimidine biosynthetic pathway at the orotidine-5′-phosphate decarboxylase level was previously demonstrated to affect riboflavin production in the industrial producer fungus Ashbya gossypii. However, the molecular basis for the unusual sensitivity to uracil displayed by the pyrimidine auxotroph A. gossypii Agura3 was unknown. Here, uridine was shown to be the only intermediate of the pyrimidine salvage pathway able to fully restore this mutant's growth. Conversely, uracil, which is routinely used to rescue pyrimidine auxotrophs, had a dose-dependent growth-inhibitory effect. Uracil phosphoribosyltransferase (UPRT) is the pyrimidine salvage pathway enzyme responsible for converting uracil to uridine monophosphate in the presence of phosphoribosyl pyrophosphate (PRPP). Characterization of the A. gossypii UPRT, as produced and purified from Escherichia coli, revealed that uracil concentrations above 1 mM negatively affected its activity, thus explaining the hypersensitivity of the Agura3 mutant to uracil. Accordingly, overexpression of the AgUPRT encoding-gene in A. gossypii Agura3 led to similar growth on rich medium containing 5 mM uracil or uridine. Decreased UPRT activity ultimately favors the preservation of PRPP, which otherwise may be directed to other pathways. In A. gossypii, increased PRPP availability promotes overproduction of riboflavin. Thus, this UPRT modulation mechanism reveals a putative means of saving precursors essential for riboflavin overproduction by this fungus. A similar uracil-mediated regulation mechanism of the UPRT activity is reported only in two protozoan parasites, whose survival depends on the availability of PRPP. Physiological evidence here discussed indicate that it may be extended to other distantly related flavinogenic fungi.

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Section: Introductionsupporting

confidence: 52%

Section: Enzyme Activity Assaysmentioning

confidence: 99%

Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii

et al. 2019

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“…Substrate acceptance of pyrimidine and purine analogues by phosphoribosyltransferases. References: a: Soysa et al (2013); b: Rasmussen et al (1986); c: Krungkrai et al (2004); d: Schimandle et al (1987); e: Keough et al (1999); f: Krenitsky et al (1969); g: Turttle and Krenitsky (1980); h: Allen et al (2002). enzymatic strategies for the preparation of modified nucleotide triphosphates, using enzymes from pentose phosphate and nucleoside salvage pathways, and their incorporation in RNA for spectroscopic (NMR, fluorescence) studies investigated (Alvarado et al, 2014;Zeiner et al, 2008).…”

Section: Monophosphatesmentioning

confidence: 98%

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

Iglesias

Lewkowicz

Médici

et al. 2015

Biotechnology Advances

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“…This could be due to substrate inhibition of uracil phosphoribosyltransferase. The enzyme from Leishmania donovani is substrate inhibited (Soysa et al, 2013). …”

Section: Enzymes Of Pyrimidine Salvage Pathwaysmentioning

confidence: 99%

Pyrimidine metabolism in schistosomes: A comparison with other parasites and the search for potential chemotherapeutic targets

Kouni

2017

Comparative Biochemistry and Physiology Part B: Biochemistry an

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Schistosomes are responsible for the parasitic disease schistosomiasis, an acute and chronic parasitic ailment that affects more than 240 million people in 70 countries worldwide. It is the second most devastating parasitic disease after malaria. At least 200,000 deaths per year are associated with the disease. In the absence of the availability of vaccines, chemotherapy is the main stay for combating schistosomiasis. The antischistosomal arsenal is currently limited to a single drug, Praziquantel, which is quite effective with a single-day treatment and virtually no host-toxicity. Recently, however, the question of reduced activity of Praziquantel has been raised. Therefore, the search for alternative antischistosomal drugs merits the study of new approaches of chemotherapy. The rational design of a drug is usually based on biochemical and physiological differences between pathogens and host. Pyrimidine metabolism is an excellent target for such studies. Schistosomes, unlike most of the host tissues, require a very active pyrimidine metabolism for the synthesis of DNA and RNA. This is essential for the production of the enormous numbers of eggs deposited daily by the parasite to which the granulomas response precipitate the pathogenesis of schistosomiasis. Furthermore, there are sufficient differences between corresponding enzymes of pyrimidine metabolism from the host and the parasite that can be exploited to design specific inhibitors or “subversive substrates” for the parasitic enzymes. Specificities of pyrimidine transport also diverge significantly between parasites and their mammalian host. This review deals with studies on pyrimidine metabolism in schistosomes and highlights the unique characteristic of this metabolism that could constitute excellent potential targets for the design of safe and effective antischistosomal drugs. In addition, pyrimidine metabolism in schistosomes is compared with that in other parasites where studies on pyrimidine metabolism have been more elaborate, in the hope of providing leads on how to identify likely chemotherapeutic targets which have not been looked at in schistosomes.

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Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs

Cited by 12 publications

References 31 publications

Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii

Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

Pyrimidine metabolism in schistosomes: A comparison with other parasites and the search for potential chemotherapeutic targets

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