Characterisation of cDNA clones for hypoxanthine–guanine phosphoribosyltransferase from the human malarial parasite,<i>Plasmodium falciparum</i>: comparisons to the mammalian gene and protein

King, Angus; Melton, David W.

doi:10.1093/nar/15.24.10469

Cited by 49 publications

(34 citation statements)

References 33 publications

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“…Also, the most prominent protein bands from the bacteria expressing the four recombinant enzymes (see Fig. 1) possess electrophoretic mobilities that are consistent with the predicted molecular weights of their subunits (3,4,11,13).…”

Section: Methodssupporting

confidence: 63%

“…Hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase [EC 2.4.2.8]; also hypoxanthine-guanine phosphoribosyltransferase or hypoxanthine-guanine-xanthine phosphoribosyltransferase) is a key purine salvage enzyme that has been studied extensively, and cDNAs encoding the human, schistosomal, malarial, and tritrichomonal HPRTs have been cloned and sequenced (3,4,11,13). In addition, active recombinant enzymes from humans, schistosomes, and tritrichomonads have previously been expressed in bacteria (3,5,6).…”

mentioning

confidence: 99%

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Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites

Eakin

Nieves-Alicea

Tosado-Acevedo

et al. 1995

Antimicrob Agents Chemother

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Expression plasmids encoding the hypoxanthine phosphoribosyltransferases (HPRTs) of Plasmodium falciparum, Schistosoma mansoni, Tritrichomonas foetus, and Homo sapiens were subcloned into genetically deficient Escherichia coli that requires complementation by the activity of a recombinant HPRT for growth on semidefined medium. Fifty-nine purine analogs were screened for their abilities to inhibit the growth of these bacteria. Several compounds that selectively altered the growth of the bacteria complemented by the malarial, schistosomal, or tritrichomonal HPRT compared with the growth of bacteria expressing the human enzyme were identified. These results demonstrate that the recombinant approach to screening compounds by complement selection in a comparative manner provides a rapid and efficient method for the identification of new lead compounds selectively targeted to the purine salvage enzymes of parasites.

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Section: Methodssupporting

confidence: 63%

mentioning

confidence: 99%

Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites

Eakin

Nieves-Alicea

Tosado-Acevedo

et al. 1995

Antimicrob Agents Chemother

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“…We thought at first that the isolation of the HGPRT-II cDNA was a cloning artifact, perhaps as a result of inefficient or incomplete processing of the nascent HGPRT-II mRNA to the mature, HGPRT-I mRNA in T. gondii. This thought was reinforced by the fact that it is the smaller HGPRT-I that is homologous to all other known HGPRTs, including its closest known relative, Plasmodium falciparum HGPRT (19,20), and that all other organisms possess only one HGPRT protein.…”

Section: Resultsmentioning

confidence: 99%

The Two Toxoplasma gondii Hypoxanthine-Guanine Phosphoribosyltransferase Isozymes Form Heterotetramers

White

Ross

Davis³

et al. 2000

Journal of Biological Chemistry

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Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I⅐HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo-and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I⅐HGPRT-II and suggests that the predominant heterotetramer consists of one HG-PRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.Encephalitis caused by the apicomplexan protozoan Toxoplasma gondii is the second leading cause of death among patients with AIDS (1). Much effort has been expended on the characterization of this parasite and on the discovery and development of new drugs to treat T. gondii infections (2, 3). A widely recognized drug target in parasitic protozoa is the enzyme hypoxanthine-guanine phosphoribosyltransferase (HG-PRT 1 ; EC 2.4.2.8) (4, 5). HGPRT catalyzes the Mg 2ϩ -dependent conversion of hypoxanthine, guanine, or xanthine and ␣-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to purine nucleotides and inorganic pyrophosphate. It is a key purine salvage enzyme in T. gondii, which cannot carry out the de novo synthesis of purine nucleotides required for growth and replication (6, 7).The cloning of T. gondii HGPRT revealed the presence of two cDNAs, differing by 147 nucleotides, that appear to result from differential splicing of the nascent transcript from the single HGPRT gene (8,9). It is the smaller 230-amino acid isozyme I (HGPRT-I), which is homologous to human HGPRT, that we (11,12) 2 and others (13) have crystallized. Enzymatically active HGPRT-I is a homotetramer (12). The larger T. gondii HGPRT isozyme, HGPRT-II, has a 49-amino acid insertion following Glu 7 (GenBank TM accession number U10083). How the three-dimensional structure of HGPRT-I is altered to accommodate the insertion in HGPRT-II, which is located at a subunit interface within the HGPRT tetramer, is not known.T. gondii HGPRT is the only HGPRT known that may exist as two isozymes. From a drug design perspective, therefore, it is important to know whether Toxoplasma expresses HG-PRT-II as a stable enzyme, and if so whether HGPRT-I or HGPRT-II (or both) is the true drug target in Toxoplasma. If both HGPRT-I and HGPRT-II are expressed in Toxoplasma, do they form homotetramers only, or are they capable of forming heterotetramers as well? If so, do they form a preferred heterotetramer ...

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“…The PfHGXPRT enzyme displays high activity for all three purine substrates, with some variation in affinity for them (K m of hypoxanthine ϭ 0.46 M, K m of guanine ϭ 0.30 M, and K m of xanthine ϭ 29 M) (49). King and Melton reported the first cloning and sequencing of PfHGXPRT (35). When expressed in E. coli (31), the recombinant enzyme was shown to differ from the human enzyme in several respects, including an increased ability to utilize allopurinol as a substrate.…”

Section: P Falciparum Phosphoribosyltransferase Enzymesmentioning

confidence: 99%

Purine Salvage Pathways in the Intraerythrocytic Malaria Parasite Plasmodium falciparum

2008

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2Malaria is caused by protozoan parasites of the genus Plasmodium. Five species of Plasmodium cause infection in humans, with the majority of lethal cases caused by Plasmodium falciparum. This species is responsible for more than 500 million clinical cases of malaria each year (59). In the past 2 decades, efforts to combat this disease have been met with the emergence of widespread resistance to most of the commonly used antimalarial drugs (68). The poor efficacy of current drugs and the lack of a promising vaccine have resulted in alarming increases in the rates of malaria morbidity and mortality worldwide, with the major toll felt in the developing world.P. falciparum is transmitted to humans via the bite of an infected female anopheline mosquito. In humans, the parasite undergoes one cycle of asexual multiplication in hepatocytes, followed by several cycles of infection and multiplication in red blood cells. Whereas the hepatocytic stage is asymptomatic, the erythrocytic stage is accompanied by the destruction of the host erythrocytes, resulting in anemia and, in the absence of treatment, death. Extensive efforts are presently under way to develop a vaccine, and advances in our understanding of the biology of the parasite and its metabolic and nutritional needs offer new routes for chemotherapy. In this regard, purine metabolism holds significant promise as a target for drug development.It has long been recognized that protozoan parasites, including Plasmodium spp., are unable to synthesize purine rings de novo (4). Consistent with this observation, the sequencing of protozoan genomes has failed to uncover any genes encoding enzymes involved in the biosynthesis of purine nucleosides or nucleobases (12). Protozoan parasites rely instead on salvage of purines from the host. The strategies used in acquiring purines vary significantly among parasite genera. This review will focus primarily on the purine salvage pathways present in the Plasmodium parasite. Recent reviews have examined the metabolic pathways for purine salvage in other protozoa (11,17,29).Initial studies on purine and pyrimidine synthesis in Plasmodium parasites were performed on erythrocytic stages of the rodent malaria species P. berghei (7,8,65), the macaque monkey malaria species P. knowlesi (46), and the avian malaria parasite P. lophurae (61,66 (46). Following on from these findings, Gutteridge and Trigg found that, in P. knowlesi, all radiolabeled purines were significantly incorporated into nucleic acids while none of the pyrimidines tested were (28). These early biochemical studies demonstrated that Plasmodium parasites lack the ability to metabolize exogenous pyrimidines and instead are entirely dependent on de novo synthesis. Conversely, Plasmodium parasites are entirely reliant upon the salvage of extracellular purines (4) and are capable of metabolizing a wide variety of exogenous purine nucleobases and nucleosides.The continuous culture of P. falciparum in serum-free media is dependent upon the supply of exogenous purines (1, 43), sug...

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Characterisation of cDNA clones for hypoxanthine–guanine phosphoribosyltransferase from the human malarial parasite,Plasmodium falciparum: comparisons to the mammalian gene and protein

Cited by 49 publications

References 33 publications

Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites

Comparative complement selection in bacteria enables screening for lead compounds targeted to a purine salvage enzyme of parasites

The Two Toxoplasma gondii Hypoxanthine-Guanine Phosphoribosyltransferase Isozymes Form Heterotetramers

Purine Salvage Pathways in the Intraerythrocytic Malaria Parasite Plasmodium falciparum

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