Molecular Characterization of the First Two Enzymes of the Pentose-phosphate Pathway of Trypanosoma brucei

ABSTRACT The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme inSalmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.

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Handling Uncertainty in Dynamic Models: The Pentose Phosphate Pathway in Trypanosoma brucei

PLoS Computational Biology ◽

10.1371/journal.pcbi.1003371 ◽

2013 ◽

Vol 9 (12) ◽

pp. e1003371 ◽

Cited By ~ 29

Author(s):

Eduard J. Kerkhoven ◽

Fiona Achcar ◽

Vincent P. Alibu ◽

Richard J. Burchmore ◽

Ian H. Gilbert ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Pentose Phosphate Pathway ◽

Dynamic Models ◽

Pentose Phosphate

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Glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum

Biochemical Journal ◽

10.1042/bj20110170 ◽

2011 ◽

Vol 436 (3) ◽

pp. 641-650 ◽

Cited By ~ 32

Author(s):

Esther Jortzik ◽

Boniface M. Mailu ◽

Janina Preuss ◽

Marina Fischer ◽

Lars Bode ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Pentose Phosphate Pathway ◽

Drug Target ◽

Pentose Phosphate ◽

Bifunctional Enzyme ◽

Functional Differences ◽

Glucose 6 Phosphate Dehydrogenase ◽

First Time ◽

Human Rbcs

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

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