A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2—an ER chaperone and member of the Trx superfamily—and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.     

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low K_m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K_m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys¹⁶⁰ of LAT1 and Cys¹¹⁰ of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl₂ with a K_i of 0.56 ± 0.11 μM. The inhibitory effect of HgCl₂ for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl₂ for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys⁸⁸ and Cys⁴³⁹, altered amino acid transport. The V_max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys⁴³⁹ residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Role of C-terminal Cys-rich Region of Phytochelatin Synthase in Tolerance to Cadmium Ion Toxicity

Phytochelatins (PCs) are Cys-rich peptides, synthesized by PC synthase in response to heavy metal ions. The C-terminal Cys-rich region of the PC synthase has homology with functional domains of metallochaperone, metallothionein and thioredoxin. To test the possibility that the C-terminal Cys-rich region of PC synthase has a role in regulating PC synthesis, we introduced point mutations into the PC synthase, replacing Cys³⁵⁸, Cys³⁵⁹ Cys³⁶³ and Cys³⁶⁶ residues with Ala. The mutant PC synthase had a lower PC synthesis ability than the wild-type enzyme. Further, oxidative conditions severely damaged mutant PC synthase whilst the wild-type enzyme suffered less damage, suggesting that the Cys-rich region of PC synthase may play an important role in anti-oxidation activity. Although the C-terminal of PC synthase is not conserved, our studies support the possibility that this region performs several important biological functions.     

Mechanism-based inactivation of thioredoxin reductase from Plasmodium falciparum by Mannich bases. Implication for cytotoxicity

Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes reduction of thioredoxin disulfide (Trx). For Plasmodium falciparum, a causative agent of tropical malaria, TrxR is an essential protein which has been validated as a drug target. The high-throughput screening of 350000 compounds has identified Mannich bases as a new class of TrxR mechanism-based inhibitors. During catalysis, TrxR conducts reducing equivalents from the NADPH-reduced flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and Cys535'-Cys540', referred to as N-terminal and C-terminal cysteine pairs. The structures of unsaturated Mannich bases suggested that they could act as bisalkylating agents leading to a macrocycle that involves both C-terminal cysteines of TrxR. To confirm this hypothesis, different Mannich bases possessing one or two electrophilic centers were synthesized and first studied in detail using glutathione as a model thiol. Michael addition of glutathione to the double bond of an unsaturated Mannich base (3a) occurs readily at physiological pH. Elimination of the amino group, promoted by base-catalyzed enolization of the ketone, is followed by addition of a second nucleophile. The intermediate formed in this reaction is an alpha,beta-unsaturated ketone that can react rapidly with a second thiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can be observed spectroscopically. The data show that it is the C-terminal Cys 535'-Cys540' pair rather than the N-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turn over its natural substrate Trx, it can reduce low M(r) electron acceptors such as methyl methanethiolsulfonate by using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases, a detailed mechanism for the inactivation of TrxR is proposed.     

Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase: INSIGHTS INTO ENDOGENOUS CYSTEINE-DEPENDENT COPPER INCORPORATION*

Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe⁵¹³ on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193–201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19–26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys⁹², which is covalently bound to His⁹⁴ via an unusual thioether linkage in the holo-form, and Cys⁵²² and Cys⁵²⁵ of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information.     

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys⁵⁹/Cys⁶⁴), and a C-terminal cysteine/selenocysteine motif (Cys⁴⁹⁷/Sec⁴⁹⁸). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO^•) to the hydroxyl radical adduct (DEPMPO/HO^•). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O. Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys⁵⁹/Cys⁶⁴ dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys⁵⁹/Cys⁶⁴ dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys⁴⁹⁷/Sec⁴⁹⁸ active site. Although the bulk of the DEPMPO/HO^• signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO^• could be generated by the enzyme in an H₂O₂-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.     

Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor

The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys¹⁷⁴, Cys²²⁶, Cys²⁹⁶ and Cys⁴⁰³ are important for the GLP-1-mediated response, whereas Cys²³⁶, Cys³²⁹, Cys³⁴¹, Cys³⁴⁷, Cys⁴³⁸, Cys⁴⁵⁸ and Cys⁴⁶² are not. Extensive deletions were made in the C-terminal tail of GLP-1R in order to determine the limit for truncation. As for other family B GPCRs, we observed a direct correlation between the length of the C-terminal tail and specific binding of ¹²⁵I-GLP-1, indicating that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function.     

Crystal Structure of the Plasmodium falciparum Thioredoxin Reductase–Thioredoxin Complex

Over the last decades, malaria parasites have been rapidly developing resistance against antimalarial drugs, which underlines the need for novel drug targets. Thioredoxin reductase (TrxR) is crucially involved in redox homeostasis and essential for Plasmodium falciparum. Here, we report the first crystal structure of P. falciparum TrxR bound to its substrate thioredoxin 1. Upon complex formation, the flexible C-terminal arm and an insertion loop of PfTrxR are rearranged, suggesting that the C-terminal arm changes its conformation during catalysis similar to human TrxR. Striking differences between P. falciparum and human TrxR are a Plasmodium-specific insertion and the conformation of the C-terminal arm, which lead to considerable differences in thioredoxin binding and disulfide reduction. Moreover, we functionally analyzed amino acid residues involved in substrate binding and in the architecture of the intersubunit cavity, which is a known binding site for disulfide reductase inhibitors. Cell biological experiments indicate that P. falciparum TrxR is indeed targeted in the parasite by specific inhibitors with antimalarial activity. Differences between P. falciparum and human TrxR and details on substrate reduction and inhibitor binding provide the first solid basis for structure-based drug development and lead optimization.     

Disruption of the PfPK7 gene impairs schizogony and sporogony in the human malaria parasite Plasmodium falciparum

PfPK7 is an orphan protein kinase of Plasmodium falciparum with maximal homology to MEK3/6 and to fungal protein kinase A proteins in its C-terminal and N-terminal regions, respectively. We showed previously that recombinant PfPK7 is active on various substrates but is unable to phosphorylate the Plasmodium falciparum mitogen-activated protein kinase homologues, suggesting that it is not a MEK functional homologue. Using a reverse genetics approach to investigate the function of this enzyme in live parasites, we now show that PfPK7⁻ parasite clones display phenotypes at two stages of their life cycle: first, a decrease in the rate of asexual growth in erythrocytes associated with a lower number of daughter merozoites generated per schizont, and second, a dramatic reduction in the ability to produce oocysts in the mosquito vector. A normal asexual growth rate and the ability to produce oocysts are restored if a functional copy of the PfPK7 gene is reintroduced into the PfPK7⁻ parasites. Hence, PfPK7 is involved in a pathway that regulates parasite proliferation and development.     

Crystal Structure of Staphylococcus aureus Metallopeptidase (Sapep) Reveals Large Domain Motions between the Manganese-bound and Apo-states

Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn²⁺-dependent dipeptidase. The crystal structure of this protein in the Mn²⁺-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys¹⁵⁵ and Cys¹⁷⁸, are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.     

The cysteine-rich domain of synaptosomal-associated protein of 23 kDa (SNAP-23) regulates its membrane association and regulated exocytosis from mast cells

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys⁻ mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR3_1–145 from multidrug resistance protein into SNAP-23 Cys⁻ mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys⁻ and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys⁻-MDR3_1–145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells.     

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

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.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ^? cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.     

Biochemical and structural characterization of the apicoplast dihydrolipoamide dehydrogenase of Plasmodium falciparum

PDC (pyruvate dehydrogenase complex) is a multi-enzyme complex comprising an E1 (pyruvate decarboxylase), an E2 (dihydrolipomide acetyltransferase) and an E3 (dihydrolipoamide dehydrogenase). PDC catalyses the decarboxylation of pyruvate and forms acetyl-CoA and NADH. In the human malaria parasite Plasmodium falciparum, the single PDC is located exclusively in the apicoplast. Plasmodium PDC is essential for parasite survival in the mosquito vector and for late liver stage development in the human host, suggesting its suitability as a target for intervention strategies against malaria. Here, PfaE3 (P. falciparum apicoplast E3) was recombinantly expressed and characterized. Biochemical parameters were comparable with those determined for E3 from other organisms. A homology model for PfaE3 reveals an extra anti-parallel β-strand at the position where human E3BP (E3-binding protein) interacts with E3; a parasite-specific feature that may be exploitable for drug discovery against PDC. To assess the biological role of Pfae3, it was deleted from P. falciparum and although the mutants are viable, they displayed a highly synchronous growth phenotype during intra-erythrocytic development. The mutants also showed changes in the expression of some mitochondrial and antioxidant proteins suggesting that deletion of Pfae3 impacts on the parasite''s metabolic function with downstream effects on the parasite''s redox homoeostasis and cell cycle.     

Ycf93 (Orf105), a Small Apicoplast-Encoded Membrane Protein in the Relict Plastid of the Malaria Parasite Plasmodium falciparum That Is Conserved in Apicomplexa

Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor shared with dinoflagellate algae. The apicoplast is a useful drug target; blocking housekeeping pathways such as genome replication and translation in the organelle kills parasites and protects against malaria. The apicoplast of Plasmodium falciparum encodes 30 proteins and a suite of rRNAs and tRNAs that facilitate their expression. orf105 is a hypothetical apicoplast gene that would encode a small protein (PfOrf105) with a predicted C-terminal transmembrane domain. We produced antisera to a predicted peptide within PfOrf105. Western blot analysis confirmed expression of orf105 and immunofluorescence localised the gene product to the apicoplast. Pforf105 encodes a membrane protein that has an apparent mass of 17.5 kDa and undergoes substantial turnover during the 48-hour asexual life cycle of the parasite in blood stages. The effect of actinonin, an antimalarial with a putative impact on post-translational modification of apicoplast proteins like PfOrf105, was examined. Unlike other drugs perturbing apicoplast housekeeping that induce delayed death, actinonin kills parasites immediately and has an identical drug exposure phenotype to the isopentenyl diphosphate synthesis blocker fosmidomycin. Open reading frames of similar size to PfOrf105, which also have predicted C-terminal trans membrane domains, occur in syntenic positions in all sequenced apicoplast genomes from Phylum Apicomplexa. We therefore propose to name these genes ycf93 (hypothetical chloroplast reading frame 93) according to plastid gene nomenclature convention for conserved proteins of unknown function.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

Fingerprinting the substrate specificity of M1 and M17 aminopeptidases of human malaria, Plasmodium falciparum

Background

Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs.

Methodology/Principal Findings

To define the substrate specificity of recombinant forms of these two malaria aminopeptidases we used a new library consisting of 61 fluorogenic substrates derived both from natural and unnatural amino acids. We obtained a detailed substrate fingerprint for recombinant forms of the enzymes revealing that PfM1AAP exhibits a very broad substrate tolerance, capable of efficiently hydrolyzing neutral and basic amino acids, while PfM17LAP has narrower substrate specificity and preferentially cleaves bulky, hydrophobic amino acids. The substrate library was also exploited to profile the activity of the native aminopeptidases in soluble cell lysates of P. falciparum malaria.

Conclusions/Significance

This data showed that PfM1AAP and PfM17LAP are responsible for majority of the aminopeptidase activity in these extracts. These studies provide specific substrate and mechanistic information important for understanding the function of these aminopeptidases and could be exploited in the design of new inhibitors to specifically target these for anti-malaria treatment.     

Structural properties of the peroxiredoxin AhpC2 from the hyperthermophilic eubacterium Aquifex aeolicus

Peroxiredoxins (Prxs) are thiol peroxidases that scavenge various peroxide substrates such as hydrogen peroxide (H₂O₂), alkyl hydroperoxides and peroxinitrite. They also function as chaperones and are involved in signal transduction by H₂O₂ in eukaryotic cells. The genome of Aquifex aeolicus, a microaerophilic, hyperthermophilic eubacterium, encodes four Prxs, among them an alkyl hydroperoxide reductase AhpC2 which was found to be closely related to archaeal 1-Cys peroxiredoxins. We determined the crystal structure of AhpC2 at 1.8?Å resolution and investigated its oligomeric state in solution by electron microscopy. AhpC2 is arranged as a toroid-shaped dodecamer instead of the typically observed decamer. The basic folding topology and the active site structure are conserved and possess a high structural similarity to other 1-Cys Prxs. However, the C-terminal region adopts an opposite orientation. AhpC2 contains three cysteines, Cys⁴⁹, Cys²¹², and Cys²¹⁸. The peroxidatic cysteine C_P⁴⁹ was found to be hyperoxidized to the sulfonic acid (SO₃H) form, while Cys²¹² forms an intra-monomer disulfide bond with Cys²¹⁸. Mutagenesis experiments indicate that Cys²¹² and Cys²¹⁸ play important roles in the oligomerization of AhpC2. Based on these structural characteristics, we proposed the catalytic mechanism of AhpC2. This study provides novel insights into the structure and reaction mechanism of 1-Cys peroxiredoxins.