Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major

Abstract

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.

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

“…Tryptophan is essential for growth of L . major [ 26 ], but these results show that the amount required (<3 μM) is very much less than the amount consumed (50 μM) when available ( S10 Fig ).…”

Phenylalanine hydroxylase: Function, structure, and regulation

“…2A), similar structure, and presumed similar catalytic mechanism (2, 3). Metazoans have three or four AAAHs‐encoding genes, but only a single gene has so far been found in protozoans, such as Leishmania major (20), and in the slime mold Dictyostelium discoideum (21). These single AAAHs have been identified as PAHs (20, 21).…”

“…Metazoans have three or four AAAHs‐encoding genes, but only a single gene has so far been found in protozoans, such as Leishmania major (20), and in the slime mold Dictyostelium discoideum (21). These single AAAHs have been identified as PAHs (20, 21). Moreover, PAH is the only AAAH found in bacteria.…”

“…The investigation of PAH in non‐mammalian organisms including bacteria readily followed the earliest studies of mammalian PAH (41). Recently, PAH has also been identified in some protozoans and slime molds, and even in nonflowering plants (20, 21, 42). While animal PAH uses BH 4 as cofactor, other natural tetrahydropterins, such as tetrahydrodictyopterin, are present in D. discoideum together with BH 4 and support the PAH function at least in vitro (21, 43).…”

“…In addition to their role in the catabolic L ‐Phe/ L ‐Tyr degradation pathway (7), PAH in lower eukaryotes has been implicated in the synthesis of (pyo)melanin, a well‐known pigment that confers advantageous properties (8–10, 20). Melanin is used by insects to encapsulate parasites (9) and by the sponge Geodia cydonium to target cells for apoptosis (8).…”