Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

Abstract

Background: Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target.

Methods: Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues.

Results: Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site.

Conclusions: Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor.

Figure 1

GCH1 reaction in the folate pathway of Plasmodium falciparum. Malaria parasites cannot salvage folate and need their own de novo folate pathway to synthesize folate derivatives [24]. GCH1 converts GTP to 7,8-dihydroneopterin 3′-triphosphate, which will become the pterin moiety of folate derivatives. Several P. falciparum strains were found to contain multiple copies of gch1 (shown here as a large blue arrow). The next step in the folate pathway of P. falciparum is driven by 6-pyruvoyltetrahydropterin synthase (PTPS) to generate 6-hydroxymethyl-7,8 dihydroneopterin (HMDHP). It is worth noting that bacteria need an extra phosphorylase enzyme to remove the phosphate groups [25]. HMDHP is activated by the addition of two phosphate groups by hydroxymethyl dihydropteridine pyrophosphokinase (HPPK). Dihydropteroate synthase (DHPS) then combines the pterin moiety with 4-aminobenzoate (pABA) to produce 7,8-dihydropteroate (DHP). The last component to be added is glutamate via the reaction driven by dihydrofolate synthase (DHFS) to form 7,8-dihydrofolate (DHF). DHF is then reduced to 5,6,7,8-tetrahydrofolate (THF) by dihydrofolate reductase (DHFR). Anti-malarial sulphadoxine (SDX) and pyrimethamine (PYR) were combined to target two enzymes in the folate pathway of malaria parasites. For the chemical detail of the malarial folate pathway, see [2].

Figure 2

Comparison of the GCH1 protein from Plasmodium falciparum to GCH1 proteins with known structures. (A) Sequence alignment of the GCH1 proteins. The GCH1 sequences were divided into the N-terminal regulatory domain and the C-terminal enzymatic core with the residue numbers on each diagram. The homology scores compared to P. falciparum GCH1were shown as percent homology and colour shade (100% and black colour to its own sequence). (B) Secondary structure diagram from known GCH1 structures and the homology model of P. falciparum GCH1 with α-helices in orange and β-strands in blue. The secondary structure diagrams of the P. falciparum GCH1 model are at the top of the alignment. The conserved amino acid residues are highlighted in black with labelled key residues (see text for detail). (C) Comparison of the overall homodecameric GCH1 structures. Two face-to-face pentameric rings are coloured in red and blue.

Figure 3

Factors affecting the activities of Plasmodium falciparum GCH1. (A) Effect of temperature shift on the GCH1 activity as shown by the production of the oxidized neopterin product. (B) Effect of salt (KCl) on the GCH1 activity. (C) Effect of pH change on the activity of P. falciparum GCH1. The pH values were varied from pH 5–13 with the data point from pH 7.8, which was chosen for the enzymatic assay. Every reaction in Figure 3A-3C was performed for 90 minutes. (D) Effect of 8-oxo-GTP on the activity of P. falciparum GCH1. The inhibitory effect on initial velocity was followed under various substrate concentrations.

Figure 4

Genetic complementation of Plasmodium falciparum GCH1 in bacteria. (A) Mutation effect on P. falciparum gch1 complementation. Wild-type P. falciparum GCH1 can rescue the loss of a bacterial strain without its own gch1 (folE in bacteria). The loss of functional P. falciparum GCH1 as in the H279S mutant abolishes the complementation activity. Naturally-occurring mutations (N88Y and R230K) were also tested for their genetic complementation activities. (B) Effect of the N-terminal truncation on genetic complementation. A series of the N-terminal truncates was made in order to test their effect on genetic complementation. (C) Effect of the C-terminal helix deletion on genetic complementation. The well-conserved C-terminal helix was removed and tested for the complementation activity by the mutant.

Figure 5

Comparison of the Plasmodium falciparum GCH1 homology model and the GCH1 structure from Thermus thermophilus. (A) and (C) the structure of T. thermophilus GCH1 showing the active site. (B) and (D) The homology model of P. falciparum GCH1 with the same views. See text for detail.