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Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase i
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Document Title
Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase i
Author
Kümpornsin K., Kotanan N., Chobson P., Kochakarn T., Jirawatcharadech P., Jaru-Ampornpan P., Yuthavong Y., Chookajorn T.
Name from Authors Collection
Affiliations
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand; Center of Excellence in Malaria, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; Mahidol Vivax Research Unit, Center of Excellence in Malaria, Mahidol University, Bangkok 10400, Thailand
Type
Article
Source Title
Malaria Journal
ISSN
14752875
Year
2014
Volume
13
Issue
1
Open Access
Gold, Green
Publisher
BioMed Central Ltd.
DOI
10.1186/1475-2875-13-150
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. © 2014 Kümpornsin et al.; licensee BioMed Central Ltd.
Keyword
Antifolate | Folate pathway | GTP cyclohydrolase I | Malaria
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
Funding Sponsor
National Science and Technology Development Agency; Thailand Research Fund
License
CC BY
Rights
Author
Publication Source
Scopus