Plasmodium falciparum antioxidant protein reveals a novel mechanism for balancing turnover and inactivation of peroxiredoxins

Abstract

Life under aerobic conditions has shaped peroxiredoxins (Prx) as ubiquitous thiol-dependent hydroperoxidases and redox sensors. Structural features that balance the catalytically active or inactive redox states of Prx, and, therefore, their hydroperoxidase or sensor function, have so far been analyzed predominantly for Prx1-type enzymes. Here we identify and characterize two modulatory residues of the Prx5-type model enzyme PfAOP from the malaria parasite Plasmodium falciparum. Gain- and loss-of-function mutants reveal a correlation between the enzyme parameters and the inactivation susceptibility of PfAOP with the size of residue 109 and the presence or absence of a catalytically relevant but nonessential cysteine residue. Based on our kinetic data and the crystal structure of PfAOP(L109M), we suggest a novel mechanism for balancing the hydroperoxidase activity and inactivation susceptibility of Prx5-type enzymes. Our study provides unexpected insights into Prx structure-function relationships and contributes to our understanding of what makes Prx good enzymes or redox sensors.

Knockout of the peroxiredoxin 5 homologue PFAOP does not affect the artemisinin susceptibility of Plasmodium falciparum

“…2a). The detected consumption of NADPH in the presence of artemisinins or di- tert -butyl peroxide (DTBP) was comparable to the background activity of negative controls without peroxide substrate, whereas the conversion of tert -butyl hydroperoxide (tBOOH) served as a positive control as described previously 45, 47 . Thus, Pf AOP does not convert endoperoxides and its enzymatic activity is restricted to hydroperoxide substrates.

Figure 2Peroxidase activity of recombinant Pf AOP in the presence of artemisinins.

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“…2c). No inhibitory effects were detected except for the positive control with tBOOH, which is not only a substrate but also an irreversible inhibitor that ‘overoxidizes’ Pf AOP 45, 47 . In summary, artemisinins are neither reversible nor irreversible inhibitors of recombinant Pf AOP and enzymatic assays do not support a direct effect of Pf AOP on artemisinins and vice versa .…”

“…Coupled enzymatic assays containing recombinant Pf AOP, Pf GR and Pf Grx were carried out at 25 °C using a thermostatted Jasco V-650 UV-visual spectrophotometer as described previously 45, 47 . All assays were performed in assay buffer containing 0.1 M Tris-HCl, 1 mM EDTA, pH 8.0.…”

“…The dual localization of Pf AOP presumably arose from a gene fusion event following a horizontal prokaryote-to-eukaryote gene transfer 41 . Although studies on recombinant Pf AOP confirmed its peroxidase activity in vitro 45–47 , its physiological relevance for hydroperoxide removal or redox homeostasis has not been addressed before.…”

Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins

“…In our roGFP2 assays we choose to use H 2 O 2 to initiate GSSG formation. Whilst yeast do not harbor any bona fide glutathione peroxidase, it was recently shown that GSH and/or Grx can reduce both 1-Cys and typical 2-Cys peroxiredoxins thereby leading to GSSG production 42,46,47,49,50 . Furthermore, GSSG was shown to readily accumulate in Δglr1 cells following H 2 O 2 treatment 45 .…”