Adenine Aminohydrolase from Leishmania donovani

Boitz, Jan M.; Strasser, Richard; Hartman, Charles U.; Jardim, Armando; Ullman, Buddy

doi:10.1074/jbc.m111.307884

Cited by 21 publications

(12 citation statements)

References 83 publications

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“…The finding that extracts prepared from the Δ aah /Δ aprt double null mutant fail to convert adenine to nucleotides indicate that AAH and APRT are the sole mechanisms by which adenine can be assimilated into the parasite nucleotide pool. Furthermore, because these incorporation assays were conducted at 20 μM [ 14 C]adenine, a concentration that exceeds the K m value of APRT by an order of magnitude [16] but is comparable to the K m value determined for AAH [10], these findings strengthen the notion that AAH is the preferred route of adenine uptake in L. donovani .…”

supporting

confidence: 60%

“…As expected, wild type, Δ aprt , and Δ aah promastigotes displayed robust growth in adenine, while Δ aah /Δ aprt promastigotes failed to proliferate (Table I). As shown previously, neither Δ adss nor Δ hgprt /Δ xprt promastigotes could grow in adenine [9,17], although when a Δ aah mutation was inserted into each of these genetic backgrounds, the cells grew robustly [10,17]. Furthermore, pharmacologic simulation of the AAH deficiency with dCF enabled Δ adss [17] and Δ hgprt /Δ xprt (Table 1) promastigotes to proliferate with adenine as the sole purine nutrient but prevented growth of Δ aprt promastigotes by blocking adenine conversion to hypoxanthine via AAH.…”

mentioning

confidence: 94%

“…The construction and phenotypic characterization of the Δ aprt and Δ aah cell lines have been described previously [10,12]. To generate the Δ aah /Δ aprt double knockout, two APRT / aprt heterozygotes, were first created within the Δ aah background using independent targeted gene replacement constructs harboring two separate drug markers to generate Δ aah/APRT / aprt -1 and Δ aah/APRT / aprt -2 heterozygotes.…”

mentioning

confidence: 99%

“…2A). The hybridization signals observed when the blots were probed with the APRT open reading frame, the APRT 5′-flanking region, or the AAH coding region corresponded to the sizes of the restriction fragments predicted from the sequences of the APRT and AAH ORFs and their adjacent 5′ and 3′ UTRs [10,13]. The homozygous gene replacements in the parasite lines harboring Δ aprt , Δ aah , and Δ aah /Δ aprt lesions were corroborated by Western blot analysis of lysates prepared from wild type, heterozygous, and homozygous knockout parasites using polyclonal antisera specific for APRT [14] and AAH [10] (Fig.…”

mentioning

confidence: 99%

“…Included in this nutritional analysis were previously isolated gene deletion mutants with known 6-aminopurine growth deficits. These mutants include L. donovani harboring genetic lesions in adenylosuccinate synthetase (ADSS), AAH and ADSS (Δ aah /Δ adss ), HGPRT and XPRT (Δ hgprt /Δ xprt ), and the triple null mutant Δ aah /Δ hgprt /Δ xprt (Table I) [9,10,17]. Nutritional assessments were performed in purine-deficient Dulbecco’s modified Eagle medium- Leishmania (DME-L) growth medium supplemented with 5% dialyzed fetal bovine serum to which either 100 μM adenine or adenosine was added.…”

mentioning

confidence: 99%

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Adenine and adenosine salvage in Leishmania donovani

Boitz

Ullman

2013

Molecular and Biochemical Parasitology

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6-aminopurine metabolism in Leishmania is unique among trypanosomatid pathogens since this genus expresses two distinct routes for adenine salvage: adenine phosphoribosyltransferase (APRT) and adenine deaminase (AAH). To evaluate the relative contributions of APRT and AAH, adenine salvage was evaluated in Δaprt, Δaah, and Δaprt/Δaah null mutants of L. donovani. The data confirm that AAH plays the dominant role in adenine metabolism in L. donovani, although either enzyme alone is sufficient for salvage. Adenosine salvage was also evaluated in a cohort of null mutants. Adenosine is also primarily converted to hypoxanthine, either intracellularly or extracellularly, but can also be phosphorylated to the nucleotide level by adenosine kinase when the predominant pathways are genetically or pharmacologically blocked. These data provide genetic verification for the relative contributions of 6-aminopurine metabolizing pathways in L. donovani and demonstrate that all of the pathways can function under appropriate conditions of genetic or pharmacologic perturbation.

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supporting

confidence: 60%

mentioning

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Adenine and adenosine salvage in Leishmania donovani

Boitz

Ullman

2013

Molecular and Biochemical Parasitology

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Purine and pyrimidine transporters of pathogenic protozoa – conduits for therapeutic agents

Campagnaro

Koning

2020

Medicinal Research Reviews

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Purines and pyrimidines are essential nutrients for any cell. Most organisms are able to synthesize their own purines and pyrimidines, but this ability was lost in protozoans that adapted to parasitism, leading to a great diversification in transporter activities in these organisms, especially for the acquisition of amino acids and nucleosides from their hosts throughout their life cycles. Many of these transporters have been shown to have sufficiently different substrate affinities from mammalian transporters, making them good carriers for therapeutic agents. In this review, we summarize the knowledge obtained on purine and pyrimidine activities identified in protozoan parasites to date and discuss their importance for the survival of these parasites and as drug carriers, as well as the perspectives of developments in the field.

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A Leishmania-specific gene upregulated at the amastigote stage is crucial for parasite survival

et al. 2018

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The morphological and biochemical alterations between the two life stages of Leishmania are governed by stage-regulated expression of several genes. Amastigote-specific genes are believed to have a role in the survival and replication of the parasite in the hostile environment of the mammalian host. Previously, we have reported the upregulated expression of A1 gene (LdA1) at the amastigote stage at RNA level. In the present study, we have further characterized LdA1, in order to understand its role in Leishmania. Sequence homology search revealed that LdA1 is unique to the Leishmania genus. Sequence- and structure-level functional annotations predicted the involvement of LdA1 in a range of biological processes critical for survival of the parasites. Western blot confirmed the upregulated expression of LdA1 at the protein level at the amastigote stage. Overexpression of LdA1 in Leishmania did not affect its growth, phenotype, or infectivity. Attempts to generate null mutants of LdA1 by homologous recombination were not successful. Repeated inability to create null mutants of LdA1 was suggestive of gene essentiality. Mutant parasites with a single allele deletion of LdA1 (LdA1) showed reduction in motility, size, and growth rate at both the life stages in vitro, which was restored following gene add-back by episomal expression of LdA1 in mutant parasites. Although LdA1 parasites were able to infect macrophages ex vivo, their capacity to survive inside macrophages was reduced significantly (P < 0.01) beyond 72 h of infection. In conclusion, LdA1 is a Leishmania-specific gene having upregulated expression at the amastigote stage and is important for survival of Leishmania parasite.

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Adenine Aminohydrolase from Leishmania donovani

Cited by 21 publications

References 83 publications

Adenine and adenosine salvage in Leishmania donovani

Adenine and adenosine salvage in Leishmania donovani

Purine and pyrimidine transporters of pathogenic protozoa – conduits for therapeutic agents

A Leishmania-specific gene upregulated at the amastigote stage is crucial for parasite survival

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