Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

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.

A pivot mutation impedes reverse evolution across an adaptive landscape for drug resistance in Plasmodium vivax

“…This has implications for the practice of molecular epidemiology: while sequencing selected SNPs in resistance determinants might be sufficient for identifying resistance alleles in settings where populations of pathogen are ‘forward’ evolving resistance, the genomic picture is likely more complicated in reverse. The results, corroborated by findings from the field, suggest that re-evolution of the susceptible phenotype (without the growth defects of the more resistant phenotypes) is likely to occur either through the introduction of susceptible migrant genotypes from elsewhere or compensatory mutations at sites other than the ones originally arising during resistance evolution [ 57 , 58 ].…”

Plasmodial enzymes in metabolic pathways as therapeutic targets and contemporary strategies to discover new antimalarial drugs: a review

“…This gene is attractive for novel antimalarial therapeutic target because it is expressed in blood-stage parasites and gtp-ch provides the rate-limiting steps in folate pathway, as shown in another microorganism (Lee et al, 2001). Years later, Kümpornsin et al (2014) studied the enzymatic activity and genetic complementation for P. falciparum GTP cyclohydrolase I (PfGCH1). Its findings indicated that this could be a new approach to antimalarial drug development, since the assay of this enzyme showed an inhibitory effect by 8-oxo-GTP, a known GTP analogue inhibitor (Kümpornsin et al, 2014).…”

“…Years later, Kümpornsin et al (2014) studied the enzymatic activity and genetic complementation for P. falciparum GTP cyclohydrolase I (PfGCH1). Its findings indicated that this could be a new approach to antimalarial drug development, since the assay of this enzyme showed an inhibitory effect by 8-oxo-GTP, a known GTP analogue inhibitor (Kümpornsin et al, 2014).…”

How to combat emerging artemisinin resistance: Lessons from “The Three Little Pigs”

“…Cultured parasite lines from Southeast Asia are often used as strains of choice for long-term in vitro drug-selection experiments due to their adaptability to drug pressure [ 10 ]. Even though the underlying mechanism is not clear, their genetic makeups allow them to evolve compensatory mechanism(s) to offset the loss of fitness incurred by deleterious drug-resistant mutations, as demonstrated in the situation of pyrimethamine resistance [ 11 – 13 ].…”