N2 - The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK) catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD), which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA), which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5’-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5’-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.
Total NAD+ and NADH concentrations (mM) and NAD+:NADH ratios for the BY4742 wild type, NAD+ salvage pathway knockout, and NAD+de novo pathway knockout yeast strains are shown in together with previously published data on NAD+ and NADH concentrations in wild type yeast.
De novo synthesis - an overview | ScienceDirect Topics
This is not surprising since deletion of QPT1, should prevent conversion of tryptophan products into NAD+ through the de novo biosynthesis pathway.
De novo synthesis of purines pdf file | bhkxahq | Flickr
(2012), The de novo and salvage pathways of GDP-mannose biosynthesis are both sufficient for the growth of bloodstream-form Trypanosoma brucei.
de novo purine biosynthetic pathwayRat Genome …
Here, we have performed lifespan, NAD+ and NADH (the reduced form of NAD+) analyses on BY4742 wild-type, NAD+ salvage pathway knockout (npt1Delta) and NAD+ de novo pathway knockout (qpt1Delta) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR).
de novo purine biosynthetic pathwayRat ..
We observed that wild-type and qpt1Delta yeast exclusively utilized extracellular nicotinic acid for NAD+ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present.
(de novo folate synthesis, intact folate salvage, ..
We observe that wild type and qpt1Δ yeast exclusively utilize extracellular nicotinic acid for NAD+ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present.
de novo pathway the reduction of ..
Again, such a finding is not surprising since deletion of NPT1 should disrupt the salvage pathway and prevent conversion of recycled and imported nicotinic acid into NAD+ through the salvage biosynthesis pathway.