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.
Highlights
The orotidine 5Ј-monophosphate decarboxylase (OMPDC) domain is at the N terminus of the gene products for all three pathogens, whereas OMPDC is at the C terminus of the mammalian UMP synthase (UMPS) (Fig. 1B)
Most previous studies on nucleotide metabolism in Leishmania have focused on the purine nucleotide pathway because Leishmania spp., like all disease-causing parasites in humans, lack the enzymes needed to synthesize purine nucleotides de novo, precipitating a great deal of interest in the purine salvage enzymes as possible antiparasitic therapeutic targets (38 – 40)
The pyrimidine biosynthetic genes in Leishmania spp. and other trypanosomatids are syntenic and clustered in a contiguous region of 25 kilobases within the genome. This grouping of genes into an operon-like cluster is unusual in Leishmania spp., considering that the genes for virtually all other metabolic pathways are scattered throughout the genome [22, 43]
Summary
Chemicals and Reagents—Oligonucleotides were purchased from Integrated DNA Technologies, Inc. (San Diego, CA). The protein was split into two fractions, one of which was concentrated to ϳ20 mg/ml as determined by the method of Bradford [26] and used without further purification, and a second that was further purified and buffer exchanged by gel filtration chromatography. This step involved purifying protein on an ACTA Explorer FPLC with a HiLoad 26/60 Superdex prep grade G200 column running 300 mM NaCl, 5 mM UMP, and 30 mM Tris-HCl, pH 7.6. For LdUMPS, protein was used at 10 mg/ml in buffer containing 300 mM NaCl, 25 mM UMP, 2 mM DTT, and 300 mM imidazole, pH 7.6. The presence of a long unit cell axis for LdUMPS limited data collection to ϳ2.9 Å to clearly
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