Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major

Lon Fye Lye, Song Ok Kang, Joshua D. Nosanchuk, Arturo Casadevall, Stephen M. Beverley

Research output: Contribution to journalArticle

9 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)58-67
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume175
Issue number1
DOIs
StatePublished - Jan 1 2011

Keywords

  • Amino acid metabolism
  • Aromatic amino acid hydroxylase
  • Tetrahydrobiopterin
  • Trypanosomatid protozoa
  • Virulence

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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