Expression and Properties of Recombinant P. Falciparum Hypoxanthine-Guanine Phosphoribosyltransferase

Keough, Dianne T.; Ng, A.; Emmerson, B. T.; Jersey, John de

doi:10.1007/978-1-4615-5381-6_141

Cited by 4 publications

(3 citation statements)

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“…The reaction was carried out using a 1.0 cm path length with a UV Visible spectrometer 1601 (Shimadzu) in the reaction buffer (0.1 M Tris, pH 8.4; 0.11 M MgCl 2 ; at 25°C). The initial velocity (V 0 [μM/min]) was calculated using Beer-Lambert law for different concentration of the purine substrate molecules: A = Δε*L*c where A is measured absorbance, L is length of cuvette, Δε is constant (5817 M -1 *cm -1 for guanine, 2283 M -1 *cm -1 for hypoxanthine and 4685 M -1 *cm -1 for xanthine [ 39 ] and c is concentration of the product formed in one minute (V 0 ). The K m , V max and k cat values were calculated using GraphPad Prism5 according to Michaelis-Menten kinetics.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery

et al. 2018

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Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

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Section: Methodsmentioning

confidence: 99%

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery

et al. 2018

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“…The A 1% 1cm is 3.84. The activity of E. coli HPRT toward hypoxanthine, guanine, and xanthine was determined spectrophotometrically (Keough et al 1998). The K i values for the nucleoside monophosphates were determined in 0.1 M Tris‐HCl, 12 mM MgCl 2 at pH 7.4 and at 25°C.…”

Section: Methodsmentioning

confidence: 99%

Crystal structures of free, IMP‐, and GMP‐bound Escherichia coli hypoxanthine phosphoribosyltransferase

et al. 2002

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Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Å resolution) and for the enzyme in complex with the reaction products, inosine 5Ј-monophosphate (IMP) and guanosine 5Ј-monophosphate (GMP) (2.8 Å resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 C␣ atoms of 1.0 Å. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg 2+ in vivo. In the free structure, a Mg 2+ is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg 2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.Keywords: Crystal structure; phosphoribosyltransferase; purine salvage; Escherichia coli; enzymology Purine nucleoside monophosphates are either synthesized de novo from simple precursors or through a salvage pathway. Most organisms, including bacteria, possess both the de novo and salvage pathways. However, some microorganisms such as the protozoan parasites do not possess the de novo pathway and therefore, depend entirely on the transport of preformed purine bases from their host cells for production of their 6-oxopurine nucleoside monophosphates (Ullman 1995). The key enzymes in the salvage pathway are the 6-oxopurine phosphoribosyltransferases (H/G/ XPRTases), which catalyze the synthesis of the purine nucleoside monophosphates inosine 5Ј-monophosphate (IMP), guanosine 5Ј-monophosphate (GMP), or xanthosine 5Ј-monophosphate (XMP) from 5-phospho-␣-D-ribosyl-1-pyrophosphate (PRib-PP) and hypoxanthine, guanine, or xanthine. A divalent cation, usually magnesium, is essential for catalysis (Musick 1981).In contrast with most eukaryotes, which have only one 6-oxopurine PRTase, enteric bacteria such as Escherichia coli possesses two, HPRT (E.C. 2.4.2.8) and XGPRT (E.C. Reprint requests to: John de Jersey, Department of Biochemistry and Molecular Biology, School of Molecular and Microbial Science, The University of Queensland, St Lucia, Qld, 4072 Australia;3,] pyra...

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“…The 6‐oxopurine PRTase of a number of organisms have been studied in depth, and in some parasites they have been identified as potential therapeutic targets owing to the absence in these organisms of a de novo purine synthesis pathway, which makes salvage pathways essential for survival. Parasites whose phosphoribosyltransferases have been investigated include Tritrichomonas foetus [11,12] , Giardia lamblia [13,14] , Plasmodium falciparum [15] , Toxoplasma gondii [16] , Schistosoma mansoni [17], and Trypanosoma cruzi [18,19] . The crystal structures of many members of the PRTase family have been resolved in both the absence and the presence of substrates, products, and transition‐state inhibitors.…”

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confidence: 99%

Bioinformatic Analysis of Helicobacter pylori XGPRTase: A Potential Therapeutic Target

Duckworth

Ménard²,

Mégraud³

et al. 2006

Helicobacter

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Expression and Properties of Recombinant P. Falciparum Hypoxanthine-Guanine Phosphoribosyltransferase

Cited by 4 publications

References 23 publications

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery

Crystal structures of free, IMP‐, and GMP‐bound Escherichia coli hypoxanthine phosphoribosyltransferase

Bioinformatic Analysis of Helicobacter pylori XGPRTase: A Potential Therapeutic Target

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