| Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major. | |
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MedLine Citation:
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PMID: 20887755 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H(4)B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H(4)B 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 H(4)B 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 H(4)B-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. |
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Authors:
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Lon-Fye Lye; Song Ok Kang; Joshua D Nosanchuk; Arturo Casadevall; Stephen M Beverley |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2010-09-29 |
Journal Detail:
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Title: Molecular and biochemical parasitology Volume: 175 ISSN: 1872-9428 ISO Abbreviation: Mol. Biochem. Parasitol. Publication Date: 2011 Jan |
Date Detail:
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Created Date: 2010-11-04 Completed Date: 2011-02-09 Revised Date: 2013-05-17 |
Medline Journal Info:
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Nlm Unique ID: 8006324 Medline TA: Mol Biochem Parasitol Country: Netherlands |
Other Details:
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Languages: eng Pagination: 58-67 Citation Subset: IM |
Copyright Information:
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Copyright © 2010 Elsevier B.V. All rights reserved. |
Affiliation:
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Department of Molecular Microbiology, Box 8230, Washington University Medicine School, 600 S. Euclid Ave., St. Louis, MO 63110, USA. |
| Data Bank Information | |
Bank Name/Acc. No.:
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GENBANK/AY273788 |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Animals Binding Sites Conserved Sequence Culture Media / chemistry DNA, Protozoan / chemistry, genetics Gene Knockout Techniques Genetic Complementation Test Hydroxylation Leishmania major / enzymology*, genetics, growth & development Leishmaniasis, Cutaneous / parasitology, pathology Mice Microbial Viability Molecular Sequence Data Phenylalanine / metabolism* Phenylalanine Hydroxylase / genetics*, metabolism* Protein Structure, Tertiary Sequence Alignment Sequence Analysis, DNA Tryptophan / metabolism Tyrosine / metabolism* Virulence |
| Grant Support | |
ID/Acronym/Agency:
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AI21903/AI/NIAID NIH HHS; AI52733/AI/NIAID NIH HHS; R01 AI021903-20/AI/NIAID NIH HHS; R01 AI052733/AI/NIAID NIH HHS; R01 AI052733-08/AI/NIAID NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Culture Media; 0/DNA, Protozoan; 55520-40-6/Tyrosine; 63-91-2/Phenylalanine; 73-22-3/Tryptophan; EC 1.14.16.1/Phenylalanine Hydroxylase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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