Publication: Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I
Issued Date
2014
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eng
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Mahidol University
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BioMed Central
Bibliographic Citation
Malaria Journal. Vol. 13, (2014), 150
Suggested Citation
Krittikorn Kümpornsin, Namfon Kotanan, Pornpimol Chobson, Theerarat Kochakarn, Piyaporn Jirawatcharadech, Peera Jaru-ampornpan, Yongyuth Yuthavong, Thanat Chookajorn Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I. Malaria Journal. Vol. 13, (2014), 150. doi:10.1186/1475-2875-13-150 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/2732
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Title
Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I
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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 ΔfolE 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.