Functional analyses of plasmodium ferredoxin Asp97Tyr mutant related to artemisinin resistance of human malaria parasites

Abstract

Mutation of Asp97Tyr in the C-terminal region of ferredoxin (PfFd) in the apicoplast of malaria parasites was recently reported to be strongly related to the parasite’s resistance to the frontline antimalarial drug, artemisinin. We previously showed that the aromatic amino acid in the C-terminal region of PfFd is important for the interaction with its electron transfer partner, Fd-NADP+ reductase (PfFNR). Here, the importance of the aromatic–aromatic interaction between PfFd and PfFNR was shown using the kinetic analysis of the electron transfer reaction of site-directed mutants of PfFNR with PfFd. Mutation of Asp97Tyr of PfFd was further shown to increase the affinity with PfFNR by the measurements of the dissociation constant (Kd) using tryptophan fluorescence titration and the Michaelis constant (Km) in the kinetic analysis with PfFNRs. Diaphorase activity of PfFNR was inhibited by D97Y PfFd at lower concentration as compared to wild-type PfFd. Ascorbate radical scavenging activity of PfFd and electron transfer activity to a heterogeneous Fd-dependent enzyme was lower with D97Y PfFd than that of wild-type PfFd. These results showed that D97Y mutant of PfFd binds to PfFNR tighter than wild-type PfFd, and thus may suppress the function of PfFNR which could be associated with the action of artemisinin.

Effect of Artemisinin on the Redox System of NADPH/FNR/Ferredoxin from Malaria Parasites

“…one-tenth of the rate of ascorbate radical reduction and one-third of the rate of electron transfer to heterologous Fd-dependent nitrite reductase in a similar system in Ref. [ 8 ]). On the other hand, the addition of DHA (200–400 µM) did not appear to increase the rate of NADPH oxidation significantly (open circles at 400 µM DHA, and inlet table), but it even decreased at higher concentration (600 µM).…”

“…Fd-concentration-dependent NADPH oxidation was observed for both wild-type PfFd and D97Y PfFd, but the activity of the D97Y mutant was roughly half of that of the wild-type PfFd, of which the tendency was similar to the previous results of the electron transfer activity from PfFd and D97Y PfFd (at 5 µM) to a heterologous Fd-dependent enzyme, cyanobacterial nitrite reductase (1.45 and 0.62 NADPH oxidation/FNR/sec., respectively, in Ref. [ 8 ]).…”

“…We have shown that Fd and FNR from Plasmodium falciparum (PfFd and PfFNR) exhibit the structure and function equivalent to those of the plant counterparts [ 6 ] and that the C-terminal region of PfFd, especially the aromatic property of His96, is important for the interaction with PfFNR [ 7 ]. Moreover, we showed that the mutation of Asp97Tyr, which is strongly related to the parasite resistance to artemisinin [ 1 , 2 ], significantly increased the affinity (in terms of K m and K d ) for PfFNR [ 7 , 8 ] and inhibited the diaphorase activity of PfFNR at a lower concentration compared to wild-type PfFd [ 8 ].…”

Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum

“…Site-directed mutagenesis to substitute PfFd C-terminal residues indicated that an aromatic residue at positions 96 and 97 increased the PfFd-PfFNR binding affinity. A recent study analysing the interaction of PfFd-D193Y and PfFNR showed mutations leading to attenuation of PfFNR function and suggested that this could be associated with the action of artemisinin [13,14]. ARPS10 has a role in apicoplast protein translation.…”

Naturally Acquired Kelch13 Mutations in Plasmodium falciparum Strains Modulate In Vitro Ring-Stage Artemisinin-Based Drug Tolerance and Parasite Survival in Response to Hyperoxia

“…However, the role of the apicoplast in ART tolerance is generating significant interest. Genetic mutations associated with ART tolerance include fd ( 32 , 73 – 75 ) and arps10 ( 32 ), which are localized to the apicoplast, and ATG18, which, although it is not localized to the apicoplast, is associated with apicoplast biogenesis ( 76 ), suggestive of an integral role of the apicoplast in ART tolerance. In comparison, fd mutations (D193Y) inserted and removed from K13 mutant and WT strains by Stokes and colleagues ( 77 ) using gene insertion techniques exhibited no apparent effect on parasite survival.…”