Purine and pyrimidine metabolism in the trypanosomatidae

Hammond, David J.; Gutteridge, W. E.

doi:10.1016/0166-6851(84)90117-8

Cited by 257 publications

(186 citation statements)

References 78 publications

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“…We suspected the unusually high amount of thymidine (39 mg/l) included in the formulation of HMI-9 [33], in addition to any contribution from the serum or SerumPlus ™ , might be competing with the drug. As trypanosomes are pyrimidine prototrophs, an external source of thymidine should be unnecessary [38][39][40]. When thymidine was omitted, cells grew normally and HSVTK-expressing clones were about ten times more sensitive to GCV (IC 50 ~ 0.5 μg/ml: data not shown), so all experiments were conducted in HMI-9 without thymidine.…”

Section: Validating Hsvtk As a Negative Marker Selectable With Gcvmentioning

confidence: 99%

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Scahill

Pastar

Cross

2008

Molecular and Biochemical Parasitology

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The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.

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Section: Validating Hsvtk As a Negative Marker Selectable With Gcvmentioning

confidence: 99%

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Scahill

Pastar

Cross

2008

Molecular and Biochemical Parasitology

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“…Nucleoside hydrolases play a key role in the purine salvage pathway of protozoan parasites. This salvage pathway is vital for these organisms because they lack a de novo purine biosynthesis pathway (5)(6)(7). Since neither nucleoside hydrolase activity, nor the encoding genes, have been found in mammals they constitute an attractive target for drug design against a variety of pathogens.…”

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confidence: 99%

Enzyme-Substrate Interactions in the Purine-specific Nucleoside Hydrolase from Trypanosoma vivax

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Holsbeke

et al. 2002

Journal of Biological Chemistry

131

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Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and are considered as targets for drug design. We previously reported the first x-ray structure of an inosine-adenosineguanosine preferring nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (1). Here we report the 2.0-Å crystal structure of the slow D10A mutant in complex with the inhibitor 3-deaza-adenosine and the 1.6-Å crystal structure of the same enzyme in complex with a genuine substrate inosine. The enzyme-substrate complex shows the substrate bound to the enzyme in a different conformation from 3-deaza-adenosine and provides a snapshot along the reaction coordinate of the enzyme-catalyzed reaction. The chemical groups on the substrate important for binding and catalysis are mapped. The 2-OH, 3-OH, and 5-OH contribute 4.6, 7.5, and 5.4 kcal/mol to k cat /K m , respectively. Specific interactions with the exocyclic groups on the purine ring are not required for catalysis. Site-directed mutagenesis indicates that the purine specificity of the IAG-NHs is imposed by a parallel aromatic stacking interaction involving Trp 83 and Trp 260. The pH profiles of k cat and k cat /K m indicate the existence of one or more proton donors, possibly involved in leaving group activation. However, mutagenesis of the active site residues around the nucleoside base and an alanine scan of a flexible loop near the active site fail to identify this general acid. The parallel aromatic stacking seems to provide the most likely alternative mechanism for leaving group activation.

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“…In most organisms 10-formyl-tetrahydrofolate is required for purine as well as mitochondrial formyl-methionyl-tRNA synthesis. However, Leishmania is auxotrophic for purines (12), and the genomes for several trypanosomatids lack identified homologs of the 10-formyl-THF-utilizing enzymes involved in purine synthesis (13)(14)(15). 5,10-CH 2 -THF is made in the cytosol by serine hydroxymethyltransferase (SHMT) and in the mitochondrion primarily through the activity of SHMT and the glycine cleavage complex (GCC).…”

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confidence: 99%

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

Scott

Hickerson

Vickers

et al. 2008

Journal of Biological Chemistry

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For the human pathogen Leishmania major, a key metabolic function is the synthesis of thymidylate, which requires 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF). 5,10-CH 2 -THF can be synthesized from glycine by the mitochondrial glycine cleavage complex (GCC). Bioinformatic analysis revealed the four subunits of the GCC in the L. major genome, and the role of the GCC in parasite metabolism and virulence was assessed through studies of the P subunit (glycine decarboxylase (GCVP)). First, a tagged GCVP protein was expressed and localized to the parasite mitochondrion. Second, a gcvP ؊ mutant was generated and shown to lack significant GCC activity using an indirect in vivo assay after incorporation of label from [2-14 C]glycine into DNA. The gcvP ؊ mutant grew poorly in the presence of excess glycine or minimal serine; these studies also established that L. major promastigotes require serine for optimal growth. Although gcvP ؊ promastigotes and amastigotes showed normal virulence in macrophage infections in vitro, both forms of the parasite showed substantially delayed replication and lesion pathology in infections of both genetically susceptible or resistant mice. These data suggest that, as the physiology of the infection site changes during the course of infection, so do the metabolic constraints on parasite replication. This conclusion has great significance to the interpretation of metabolic requirements for virulence. Last, these studies call attention in trypanosomatid protozoa to the key metabolic intermediate 5,10-CH 2 -THF, situated at the junction of serine, glycine, and thymidylate metabolism. Notably, genome-based predictions suggest the related parasite Trypanosoma brucei is totally dependent on the GCC for 5,10-CH 2 -THF synthesis.

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Purine and pyrimidine metabolism in the trypanosomatidae

Cited by 257 publications

References 78 publications

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Enzyme-Substrate Interactions in the Purine-specific Nucleoside Hydrolase from Trypanosoma vivax

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

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