Plasmodium falciparum Possesses a Classical Glutaredoxin and a Second, Glutaredoxin-like Protein with a PICOT Homology Domain

275

233

Glutaredoxins catalyze glutathione-dependent thiol disulfide oxidoreductions via a GSH-binding site and active cysteines. Recently a second human glutaredoxin (Grx2), which is targeted to either mitochondria or the nucleus, was cloned. Grx2 contains the active site sequence CSYC, which is different from the conserved CPYC motif present in the cytosolic Grx1. Here we have compared the activity of Grx2 and Grx1 using glutathionylated substrates and active site mutants. The kinetic studies showed that Grx2 catalyzes the reduction of glutathionylated substrates with a lower rate but higher affinity compared with Grx1, resulting in almost identical catalytic efficiencies (k cat /K m ). Permutation of the active site motifs of Grx1 and Grx2 revealed that the CSYC sequence of Grx2 is a prerequisite for its high affinity toward glutathionylated proteins, which comes at the price of lower k cat . Furthermore Grx2 was a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates. Using this novel electron donor pathway, Grx2 reduced low molecular weight disulfides such as CoA but with particular high efficiency glutathionylated substrates including GSSG. These results suggest an important role for Grx2 in protection and recovery from oxidative stress.

Section: Dual Electron Donor Specificity Of Human Glutaredoxinmentioning

confidence: 72%

Human Mitochondrial Glutaredoxin Reduces S-Glutathionylated Proteins with High Affinity Accepting Electrons from Either Glutathione or Thioredoxin Reductase

Johansson

Lillig

Holmgren

2004

275

233

“…P. falciparum TrxR (2-10 milliunits/ml), 100 M NADPH, and FP were added, and the reaction was initiated with 20 M PfTrx and followed at 340 nm. Glutaredoxin Assays-Grx activity was measured in the bis(2-hydroxyethyl)-disulfide (HEDS) assay at 340 nm as described previously (15). Corrections were made for the spontaneous reactions between HEDS and GSH and between FP and other assay components (GSHdependent FP degradation, the reaction between FP and GR and the reaction between FP and NADPH).…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Heme-interacting Proteins in the Malaria Parasite, Plasmodium falciparum

Campanale

Nickel²,

Daubenberger

et al. 2003

Self Cite

The degradation of hemoglobin by the malaria parasite, Plasmodium falciparum, produces free ferriprotoporphyrin IX (FP) as a toxic by-product. In the presence of FP-binding drugs such as chloroquine, FP detoxification is inhibited, and the build-up of free FP is thought to be a key mechanism in parasite killing. In an effort to identify parasite proteins that might interact preferentially with FP, we have used a mass spectrometry approach. Proteins that bind to FP immobilized on agarose include P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), P. falciparum glutathione reductase (PfGR), and P. falciparum protein disulfide isomerase. To examine the potential consequences of FP binding, we have examined the ability of FP to inhibit the activities of GAPDH and GR from P. falciparum and other sources. FP inhibits the enzymic activity of Pf-GAPDH with a K i value of 0.2 M, whereas red blood cell GAPDH is much less sensitive. By contrast, PfGR is more resistant to FP inhibition (K i > 25 M) than its human counterpart. We also examined the ability of FP to inhibit the activities of the additional antioxidant enzymes, P. falciparum thioredoxin reductase, which exhibits a K i value of 1 M, and P. falciparum glutaredoxin, which shows more moderate sensitivity to FP. The exquisite sensitivity of PfGAPDH to FP may indicate that the glycolytic pathway of the parasite is particularly susceptible to modulation by FP stress. Inhibition of this pathway may drive flux through the pentose phosphate pathway ensuring sufficient production of reducing equivalents to counteract the oxidative stress induced by FP build-up.

“…The glutaredoxin assay system essentially contained the same components as described above with the exception that 1-10 M P. falciparum glutaredoxin, prepared according to Ref. 32, was included into the assay before the reaction was initiated by addition of H 2 O 2 . In the third assay system it was assessed whether lipoic acid acts as a reductant for both parasite 1-Cys peroxiredoxins using the following assay conditions, 200 M NADH, 5-50 M lipoamide, 5 units of dihydrolipoamide dehydrogenase (Sigma), 0.5-20 M peroxiredoxin, and 100 M H 2 O 2 .…”

Section: Methodsmentioning

confidence: 99%

Peroxiredoxin-linked Detoxification of Hydroperoxides in Toxoplasma gondii

Akerman

Müller

2005

The apicomplexan parasite Toxoplasma gondii is highly susceptible to oxidative stress caused by tertbutyl-hydroperoxide, juglone, and phenazine methylsulfate with IC 50 in the nanomolar range. Using dichlorofluorescein diacetate, a detector of endogenous oxidative stress, it was shown that juglone and phenazine methylsulfate are potentially toxic to the parasites without affecting the host cells. These results demonstrate that T. gondii is vulnerable to oxidative challenge that results from disruption of its redox balance and so this could be an effective approach to therapeutic intervention. This study has characterized redox active and antioxidant peroxidases belonging to the class of 1-Cys and 2-Cys peroxiredoxins that play crucial roles in maintaining redox balance. The tachyzoite stages of T. ؊1 s ؊1 is unusually high, which qualifies the enzyme as an extremely potent antioxidant. Kinetic analyses revealed that TgTrx-Px1 is inhibited by tert-butyl hydroperoxide, and apparent inhibition constants were determined to be between 33 and 35.6 M depending on the concentration of the non-inhibitory substrate thioredoxin. TgTrx-Px2 protected glutamine synthetase from inactivation by Fe 3؉ /DTT, showing that it is an active peroxiredoxin.