Gene links: No gene identifiers were provided.

Description:

Phosphofructokinase (PFK) is a glycolytic enzyme located in the glycosomes of trypanosomatids. Two distantly related classes of PFK can be distinguished in nature, those that use ATP as phospho donor, and those that use PPi.

Validation Glycolysis is essential for bloodstream-form trypanosomes [reviewed in 1]. By a large number of experiments, involving enzyme inhibitors and RNAi, it has been shown that approximately 50% reduction of the glycolytic flux is sufficient to kill the parasites [2,3]. The contribution of PFK to the control of the glycolytic flux in bloodstream-form T. brucei has been determined by RNAi; partial depletion of the enzyme activity was shown to be sufficient to kill the parasites [2].

Description Enzyme Properties. The kinetic properties of PFK, purified from bloodstream-form trypanosomes, have been studied in great detail [4,5]. Considerable differences from mammalian PFKs were found; most strikingly, the activity of the enzyme is not regulated by most of the effectors that regulate PFK in other organisms. The enzyme in T. brucei is encoded by a single gene [6]. It has been expressed in Escherichia coli, purified, kinetically characterized and crystallized [7,8]. Crystal Structure. The apo crystal structure has been determined [9; PDB accession code 2HIG], as well as the holo structure with ATP (10; PDB accession code 3F5M). No crystal structure of human PFK or any eukaryote PFK other than T. brucei PFK is known as yet. The trypanosomatid PFKs, despite their absolute dependence on ATP as phospho donor, cluster phylogenetically with PPi-dependent PFKs which are very different from the ATP-PFKs found in all vertebrates, yeast and many protozoa and bacteria [6]. Comparison of the structures of T. brucei PFK with bacterial ATP-PFKs showed conservation of the overall fold, yet considerable differences in various parts of the enzyme including the active site. Sequence comparisons with human PFK (21% identity), and structure modelling showed that these differences must also occur between the trypanosome and its human homologue and renders the trypanosome enzyme a prime drug target candidate [9]. Drug Discovery. Preliminary research involving database mining and synthesis founded on structure-based design and combinatorial chemistry has yielded parasite-enzyme selective active-site inhibitors with IC50 values in the low micromolar range [11]. Some of these inhibitors kill cultured bloodstream-form trypanosomes but cultured human cells (MCR-5 fibroblasts) only at higher concentrations.

1. Verlinde et al, Drug Resistance Updates (2001) 4, 50-65.
2. Albert et al, J. Biol. Chem. (2005) 280, 28306-28315.
3. Haanstra et al, Proc. 11th Int. Congr. Parasitol. Glasgow (ISBN 88-7587-273-2), (2006) pp. 91-96.
4. Ngwagwu and Opperdoes, Acta Trop. (1982) 61-72.
5. Cronin and Tipton, Biochem. J. (1985) 227, 113-124.
6. Michels et al, Eur. J. Biochem. (1997) 250, 698-704.
7. Claustre et al, Biochemistry (2002) 41, 10183-10193.
8. Keillor et al, Acta Crystallogr D (2003) 59, 532-534.
9. Martinez et al, J. Mol. Biol. (2007) 366, 1185-1198.
10. McNae et al, J. Mol. Biol. (2008) in press.
11. Nowicki et al, Bioorg. & Med. Chem. (2008) 16, 5050-5051.

Target ID: geneDB number = Tb927.3.3270 EC number (if enzyme) = EC 2.7.1.11 target name = PFK

Validation: Genetic OR chemical evidence

Assay status: Assay exists but not yet in microtitre plate format

Availability: Can be expressed > 1 mg/L in soluble, active form

Activity: Significant (>= 1 full time person)

No references provided.