Antimalarial pantothenamide metabolites target acetyl-coenzyme A biosynthesis in Plasmodium falciparum.

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl-coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl-coenzyme A synthetase and acyl-coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.

Kinetic and thermodynamic features reveal that Escherichia coli BCP is an unusually versatile peroxiredoxin.

In Escherichia coli, bacterioferritin comigratory protein (BCP) is a peroxiredoxin (Prx) that catalyzes the reduction of H(2)O(2) and organic hydroperoxides. This protein, along with plant PrxQ, is a founding member of one of the least studied subfamilies of Prxs. Recent structural data have suggested that proteins in the BCP/PrxQ group can exist as monomers or dimers; we report here that, by analytical ultracentrifugation, both oxidized and reduced E. coli BCP behave as monomers in solution at concentrations as high as 200 µM. Unexpectedly, thioredoxin (Trx1)-dependent peroxidase assays conducted by stopped-flow spectroscopy demonstrated that V(max,app) increases with increasing Trx1 concentrations, indicating a nonsaturable interaction (K(m) > 100 µM). At a physiologically reasonable Trx1 concentration of 10 µM, the apparent K(m) value for H(2)O(2) is ~80 µM, and overall, the V(max)/K(m) for H(2)O(2), which remains constant at the various Trx1 concentrations (consistent with a ping-pong mechanism), is ~1.3 × 10(4) M(-1) s(-1). Our kinetic analyses demonstrated that BCP can utilize a variety of reducing substrates, including Trx1, Trx2, Grx1, and Grx3. BCP exhibited a high redox potential of -145.9 ± 3.2 mV, the highest to date observed for a Prx. Moreover, BCP exhibited a broad peroxide specificity, with comparable rates for H(2)O(2) and cumene hydroperoxide. We determined a pK(a) of ~5.8 for the peroxidatic cysteine (Cys45) using both spectroscopic and activity titration data. These findings support an important role for BCP in interacting with multiple substrates and remaining active under highly oxidizing cellular conditions, potentially serving as a defense enzyme of last resort.

Engineering of fluorescent reporters into redox domains to monitor electron transfers.

The rate of electron transfer through multicomponent redox systems is often monitored by following the absorbance change due to the oxidation of the upstream pyridine nucleotide electron donor (NADPH or NADH) that initiates the process. Such coupled assay systems are powerful, but because of problems regarding the rate-limiting step, they sometimes limit the kinetic information that can be obtained about individual components. For peroxiredoxins, such assays have led to widespread underestimates of their catalytic power. We show here how this problem can be addressed by a protein engineering strategy inspired by some bacterial and eukaryotic thioredoxins for which a significant fluorescence signal is generated during oxidation that provides a highly sensitive tool to directly measure electron transfers into and out of these domains. For the N-terminal domain of AhpF (a flavoprotein disulfide reductase) and Escherichia coli glutaredoxin 1, two cases not having such fluorescence signals, we have successfully added "sensor" tryptophan residues using the positions of tryptophan residues in thioredoxins as a guide. In another thioredoxin-fold redox protein, the bacterial peroxiredoxin AhpC, we used chemical modification to introduce a disulfide-bonded fluorophore. This modified AhpC still serves as an excellent substrate for the upstream AhpF electron donor but now generates a strong fluorescence signal during electron transfer. These tools have fundamentally changed our understanding of the catalytic power of peroxiredoxin systems and should also be widely applicable for improving quantitative assay capabilities in other electron transfer systems.