Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major

Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H4B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H4B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H4B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah− null mutant and an episomal complemented overexpressing derivative (pah−/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah− parasites showed reactivity with an antibody to melanin when grown with l-3,4-dihydroxyphenylalanine (l-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah− showed an increased sensitivity to Tyr deprivation, while the pah−/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah− showed no alterations in H4B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.

Graphical abstractWe establish that Leishmania major encodes an active phenylalanine hydroxylase (PAH). Genetic deletions show it may play a role in mitigating but not alleviating tyrosine auxotrophy, but that PAH is not essential for growth nor infectivity to macrophages or mice. While the only known biopterin-utilizing enzyme of Leishmania, these data argue that PAH is unlikely to account for why biopterin is an essential growth factor in trypanosomatids.Figure optionsDownload full-size imageDownload high-quality image (25 K)Download as PowerPoint slideResearch highlights▶ The Leishmania genome has a single aromatic amino acid (phenylalanine) hydroxylase. ▶L. major PAH null mutants (pah−) were viable. ▶ WT but not pah− could metabolize radiolabeled phenylalanine to tyrosine. ▶ pah− mutants were normal in metacyclogenesis and animal infectivity. ▶ PAH is unlikely to account for the Leishmania biopterin requirement.