Abstract
BackgroundAntifolates 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.MethodsPlasmodium 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.ResultsKinetic 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.ConclusionsFunctional 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.
Highlights
Antifolates are currently in clinical use for malaria preventive therapy and treatment
The rise in genomic analyses of malaria parasites revealed a unique role of GTP cyclohydrolase I (GCH1), the first and the rate-limiting enzyme of the folate pathway, in pyrimethamine resistance (Figure 1) [15]
Characteristics of Plasmodium falciparum GCH1 Plasmodium falciparum PFL1155w (PF3D7_1224000) was shown to be malarial GTP cyclohydrolase I based on enzyme kinetics and complementation studies [19,35,36]
Summary
Plasmid construction A series of P. falciparum GCH1 truncations was constructed by PCR cloning from pET45b(+)/GCH1 with Pfu DNA polymerase (Vivantis) and confirmed by direct sequencing [19]. Growth analysis was performed with preculture in LB broth (Bio Basic) supplemented with 300 μM thymidine (Sigma-Aldrich), 30 μg ml-1 kanamycin (Bio Basic) and Figure 1 GCH1 reaction in the folate pathway of Plasmodium falciparum. GCH1 converts GTP to 7,8-dihydroneopterin 3′-triphosphate, which will become the pterin moiety of folate derivatives. The step in the folate pathway of P. falciparum is driven by 6-pyruvoyltetrahydropterin synthase (PTPS) to generate 6-hydroxymethyl-7,8 dihydroneopterin (HMDHP). Protein expression and enzymatic assay Plasmodium falciparum Δ1-195 GCH1 or core GCH1 was cloned into pET45b(+) and expressed in E. coli BL21(DE3) RIL with 0.4 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for two hours. The complete reaction was composed of 50 mM Tris–HCl pH 7.8, 100 mM KCl, 20% glycerol, 250 μM GTP and 2.5 μM recombinant P. falciparum GCH1. Secondary structure data were obtained from PDB accession number 1N3T, 1WUR, 1IS8 and 1FB1
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