Document Detail


Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major.
MedLine Citation:
PMID:  20887755     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Lon-Fye Lye; Song Ok Kang; Joshua D Nosanchuk; Arturo Casadevall; Stephen M Beverley
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2010-09-29
Journal Detail:
Title:  Molecular and biochemical parasitology     Volume:  175     ISSN:  1872-9428     ISO Abbreviation:  Mol. Biochem. Parasitol.     Publication Date:  2011 Jan 
Date Detail:
Created Date:  2010-11-04     Completed Date:  2011-02-09     Revised Date:  2014-09-10    
Medline Journal Info:
Nlm Unique ID:  8006324     Medline TA:  Mol Biochem Parasitol     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  58-67     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier B.V. All rights reserved.
Data Bank Information
Bank Name/Acc. No.:
GENBANK/AY273788
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MeSH Terms
Descriptor/Qualifier:
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:
AI21903/AI/NIAID NIH HHS; AI52733/AI/NIAID NIH HHS; R01 AI021903/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:
0/Culture Media; 0/DNA, Protozoan; 42HK56048U/Tyrosine; 47E5O17Y3R/Phenylalanine; 8DUH1N11BX/Tryptophan; EC 1.14.16.1/Phenylalanine Hydroxylase
Comments/Corrections

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