# Comparative metabolism of conjugated and unconjugated pterins in Crithidia, Leishmania and African trypanosomes

## TL;DR

The study compares how three types of parasites use different forms of pterins for growth, revealing that they convert pterins to tetrahydro forms and prefer specific structures.

## Contribution

The study identifies structural requirements of pterins for growth in kinetoplastid parasites and reveals that pterins are reduced to tetrahydro forms without further conversion.

## Key findings

- T. brucei and C. fasciculata prefer 6-biopterin, while L. major strongly prefers reduced pterins.
- Pterins supporting growth are metabolized to tetrahydro forms with no evidence of further interconversion.
- Folate or dihydrofolate can partially replace unconjugated pterins in some cases.

## Abstract

Mammalian cells synthesise tetrahydrobiopterin de novo, an essential cofactor for hydroxylation of aromatic amino acids, cleavage of ether lipids and the synthesis of nitric oxide. In contrast, kinetoplastid parasites are pterin auxotrophs and none of the above metabolic functions can account for the essential requirement of an unconjugated pterin for growth. Here we investigate the pterin requirements for growth and survival of two medically important parasites (T. brucei and L. major) in comparison with the model insect parasite, Crithidia fasciculata. The pterin concentration required to support 50% of maximum growth of each parasite was determined in defined pterin-free media for a variety of naturally occurring pterins. T. brucei and C. fasciculata showed an identical order of preference with the most active being 6-biopterin, followed by dihydrobiopterin > tetrahydrobiopterin > L-neopterin > sepiapterin. In contrast, L. major showed a pronounced growth preference (>200-fold) for the reduced pterins over the the oxidised forms 6-biopterin and L-neopterin. The unnatural isomers 7-biopterin or D-neopterin supported growth poorly, or not at all, in these organisms. Other pterins were inactive. HPLC analysis of pterins supporting growth established that these were metabolised to the tetrahydro-forms (>95%) with no evidence of further interconversion. In the absence of pterins, the parasites failed to grow and lost viability with <1% surviving beyond 5–14 days. Relatively high concentrations of folate or dihydrofolate (>500 nM) could support growth in the absence of unconjugated pterin and HPLC analysis identified pteridoxamine and 6-hydroxymethylpterin (as tetrahydro-form) in cell extracts. A common feature of pterins that support growth is the presence of at least one or more linear carbon substituents at position 6 of the pteridine ring with at least one hydroxyl group, ideally in the 1S configuration. The possible essential roles of these important metabolites are discussed.

Over 75 years have passed since the discovery of “ Crithidia factor” – biopterin – as an essential cofactor involved in several important metabolic functions in mammalian cells. Despite established roles of biopterin as a cofactor in the hydroxylation of aromatic amino acids, the cleavage of glyceryl ether lipids and in the synthesis of nitric oxide in mammalian cells, these metabolic functions either do not exist in kinetoplastids or are unable to account for the essential growth requirements for pterins in these parasites. Here, we have determined structural requirements of pterin analogues that can support growth and survival of three important genera in the order Kinetoplastida. Some quantitative differences are noted in pterin preferences between C. fasciculata and T. brucei, and those of L. major. We find that the sole fate of intracellular pterins in these parasites is reduction to the corresponding tetrahydro intermediate. Folic acid can spare the growth requirement for an unconjugated pterin possibly due to trace amounts being converted to 6-hydroxymethylpterin. Future research directions for elucidation of the essential functions of pterins in these trypanosomatid parasites are discussed.