The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization

Abstract

The final two steps of de novo uridine 5-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.

Genetic Dissection of Pyrimidine Biosynthesis and Salvage in Leishmania donovani

“…The pRP-M vector expresses transgenes from the ribosomal RNA (RRNA) promoter upon integration into the RRNA array and has been used for complementation of ⌬umps parasites (40).…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Orotate phospho-ribosyltransferase, a typical type I phosphoribosyltransferase, contains the "usual" structural elements, a five-stranded ␤-sheet surrounded by a number of helices, usually four, a hood structure involved in the binding of the substrate orotate, and a flexible catalytic loop, which closes the active site on catalysis. The structures of orotate phosphoribosyltransferase from a variety of sources, including S. enterica (223), E. coli (224), S. mutans (225), Plasmodium falciparum (226), and L. donovani (227) have been published.…”

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

“…Gene sequencing, supported by biochemical studies, has revealed a number of significant differences between the pyrimidine biosynthetic pathways of Leishmania and mammals: 1) the genes encoding the pyrimidine pathway of Leishmania are syntenic (2-5), whereas the mammalian pyrimidine genes are not; 2) the genes encoding the first three enzymes in Leishmania are dis-crete, unlike the mammalian pathway in which there is a single gene encoding a trifunctional protein (6, 7); 3) the last two enzymes of the pyrimidine biosynthetic pathway are expressed as a single bifunctional protein, designated UMP synthase (UMPS), although the domain order in Leishmania and mammalian cells is reversed (3,8); and 4) the UMP synthase of Leishmania is localized within the glycosome (3), a unique peroxisomal-like organelle that is found uniquely among Leishmania and related parasites (9,10). Genetic ablation of either carbamoyl phosphate synthetase (CPS), the first enzyme of pyrimidine biosynthesis, or UMPS in L. donovani confer pyrimidine auxotrophy that can be circumvented by supplementation of the defined growth medium with uracil, uridine, deoxyuridine, cytidine, or deoxycytidine (3,5). Furthermore, both the ⌬cps and the ⌬umps null mutants exhibit a striking collateral supersensitivity to uracil, which is innocuous to wild type parasites, that is not observed with any of the ribonucleosides (5).…”

“…Parasite Cell Culture-The creation and characterization of ⌬uprt, ⌬cps, and ⌬umps L. donovani lines have been reported previously (3,5). Wild type and null mutant promastigotes were continuously cultured in 26°C in pH 7.4 DME-L medium that was supplemented with 10% Serum Plus (SAFC Biosciences, Lenexa, KS), 1 mg/ml hemin, and 100 M hypoxanthine as a purine source.…”