Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase

Téllez-Sanz, Ramiro; Yassin, Zeyad; Bernier-Villamor, Vı́ctor; Ortiz‐Salmerón, Emilia; Musso-Buendia, Juan Alexander; Barón, Carmen; Ruíz-Pérez, Luis M.; González-Pacanowska, Dolores; Garcı́a-Fuentes, Luis

doi:10.1016/j.biochi.2007.03.007

Cited by 8 publications

(4 citation statements)

References 30 publications

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“…In the absence of the metal fluorides the affinity of TbdUTPase for the non-hydrolysable substrate analogue dUpNHpp (K d = 0.43 μM), dUMP (K d = 8.1 μM) and dU (K d ∼500 μM) was comparable with that observed with other Tritryp dUTPases (TcdUTPase dUTP K d without Mg 2 + = 384 μM, dUMP + Mg 2 + = 24 μM [43]; LmdUTPase dUpNpp = 0.8 μM, dUMP = 7.3 μM, dU>500 μM [18]) ( Figure 3 and Table 2). The presence of AlF 3 and MgF 3 − had little effect on dU binding ( Figure 3A).…”

Section: Figure 2 31 P-nmr Spectra Of Products Following Reaction In supporting

confidence: 68%

On the catalytic mechanism of dimeric dUTPases

Hemsworth

González-Pacanowska

Wilson

2013

Biochemical Journal

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The Tritryps Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani are responsible for great morbidity and mortality in developing countries. Their dimeric dUTPases are members of the all-α NTP pyrophosphohydrolase family and represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared with the trimeric human enzyme. In the present paper we describe the structure of the T. brucei enzyme in open and closed conformations. Furthermore, we probe the reaction mechanism through the binding of transition state mimics both in solution and in the crystal. 31P-NMR and tryptophan fluorescence quenching in the presence of AlF3 and MgF3- identified which phosphate is subject to nucleophilic attack by a water molecule. The structures in complex with two transition state analogues confirm that the nucleophilic attack occurs on the β-phosphate in contrast with the α-phosphate in the trimeric enzymes. These results establish the structural basis of catalysis of these important housekeeping enzymes and has ramifications for the wider all-α NTP pyrophosphohydrolase family.

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Section: Figure 2 31 P-nmr Spectra Of Products Following Reaction In supporting

confidence: 68%

On the catalytic mechanism of dimeric dUTPases

Hemsworth

González-Pacanowska

Wilson

2013

Biochemical Journal

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“…Metals are clearly important for substrate binding (17), supported by the tryptophan fluorescence quenching experiments on the L. major enzyme (Fig. 4) and data reported previously (34). In the C. jejuni dUpNp complex (19), one of the water molecules coordinating the central Mg 2ϩ ion was positioned in line with the scissile bond, leading to the suggestion that this was a likely nucleophile with the reaction proceeding by a similar two ion mechanism to that proposed for the phosphoryl transfer reactions performed by DNA polymerases and many other enzymes (20,21).…”

Section: Discussionsupporting

confidence: 55%

The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine

Hemsworth

Moroz

Fogg

et al. 2011

Journal of Biological Chemistry

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Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all ␣-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric ␤-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead antitrypanosomal compounds.Various members of the genus Leishmania cause leishmaniasis, which threatens ϳ350 million people worldwide and gives rise to about two million clinical cases each year, of which ϳ25% are of the fatal visceral form (1). The disease is largely endemic to developing countries, and current treatments are expensive and can result in undesirable side effects for the patient (1). This, combined with the increasing drug resistance that is developing in the Leishmania species, means that new and novel anti-parasitic drug targets are urgently required.Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) 2 represents such a target. dUTPases catalyze the hydrolysis of dUTP to dUMP and pyrophosphate (2). This provides the starting material for the synthesis of dTMP by thymidylate synthase and in addition maintains the ratio of dTTP:dUTP in the cell at a high enough level to prevent excessive misincorporation of dUMP into the genome during DNA replication (3). dUTPase activity is essential as was shown by gene knock-outs in Escherichia coli and Saccharomyces cerevisiae. Inhibition of dUTPase activity results in futile cycles of DNA repair that ultimately cause the fragmentation of DNA and cell death (4, 5). dUTPase activity also appears to be essential in L. major (6) and the related parasite Trypanosoma brucei, where an RNAi approach was used to knock down dUTPase expression, resulting in decreased cell proliferation and growth in both procyclic and bloodstream forms of the organism (7). dUTPases have been characterized extensively both biochemically and structurally in many species. Most organisms, including humans, have trimeric dUTPases with structures largely consisting of ␤-pleated sheet (8). Three active sites are formed at the trimer interfaces by...

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“…The catalytic role of Asp80 residue of TcdUTPase was demonstrated by an Asp80Ala mutation in TcdUTPase, which altered its capability to distinguish between dUTP and dUMP while retaining the proper active site orientation, where dUMP and dUTP exhibited a non-cooperative mode of binding to the Asp80Ala mutant. This study showed the catalytic role of Asp80 residue of TcdUTPase, helpful in the rational drug designing process [ 63 ]. Crystal structure of L. major dUTPase (LmdUTPase), a crucial enzyme of the pyrimidine salvage pathway has been reported with product (UMP) bound to it.…”

Section: Enzymes Of the Pyrimidine Pathway Of Trypanosomatidsmentioning

confidence: 99%

Fresh insights into the pyrimidine metabolism in the trypanosomatids

Tiwari

Dubey

2018

Parasites Vectors

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The trypanosomatid parasites continue their killing spree resulting in significant annual mortality due to the lack of effective treatments and the prominence of these diseases in poorer countries. These dimorphic parasites thrive unchecked in the host system, outsmarting the immune mechanisms. An understanding of biology of these parasitic forms will help in the management and elimination of these fatal diseases. Investigation of various metabolic pathways in these parasites has shed light in the understanding of the unique biology of the trypansomatids. An understanding of these pathways have helped in tracing the soft targets in the metabolic pathways, which could be used as effective drug targets which would further impact the therupeutic implications. Pyrimidine pathway is a vital metabolic pathway which yields in the formation of pyrimidines, which are then integrated in nucleic acids (DNA and RNA) in sugars (UDP sugars) and lipids (CDP lipids). A wealth of data and information has been generated in the past decades by in-depth analyses of pyrimidine pathway in the trypanosomatid parasites, which can aid in the identification of anomalies between the parasitic and host counterpart which could be further harnessed to develop therapeutic interventions for the treatment of parasitic diseases. This review presents an updated and comprehensive detailing of the pyrimidine metabolism in the trypansomatids, their uniqueness and their distinctions, and its possible outcomes that would aid in the eradication of these parasitic diseases.

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Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase

Cited by 8 publications

References 30 publications

On the catalytic mechanism of dimeric dUTPases

On the catalytic mechanism of dimeric dUTPases

The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine

Fresh insights into the pyrimidine metabolism in the trypanosomatids

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