Intersubunit Interactions in Plasmodium falciparumThioredoxin Reductase

Abstract

The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M r and the low M r proteins. The well characterized Escherichia coli TrxR represents a member of the low M r class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M r proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M r TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.Infection with Plasmodium falciparum, the causative agent of malaria tropica, is responsible for 2-3 million deaths per year. The malaria parasite spends part of its developmental life cycle in human erythrocytes where it is challenged with enhanced oxidative stress. Therefore the parasite needs efficient anti-oxidants to protect itself against damages, such as nucleic acid modifications, lipid peroxidation, or oxidation of thiolcontaining proteins, caused by reactive oxygen species. The thioredoxin redox system, composed of the NADPH-dependent homodimeric thioredoxin reductase (TrxR) 1 and the 12-kDa protein thioredoxin (Trx) confers reduction of protein disulfides, ribonucleotide reductase being the most prominent example (1). Apart from this, thioredoxin interacts with a number of transcription factors in prokaryotic and eukaryotic cells, resulting in modified DNA binding activities and altered gene transcription (2). Another important function is the interaction of reduced thioredoxin with thioredoxin-dependent peroxidases, which detoxify reactive oxyg...

Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism

“…The authors (15) suggested that the mobile selenenylsulfide containing a C-terminal tail not only serves as a third redox active group, but it also blocks oxidized glutathione from binding to the enzyme. In addition, the structure confirmed the obligatory ''head-to-tail'' arrangement of high-M r TrxR, with the redox-active C-terminal tail of one subunit interacting with the active site of the opposing subunit (36,37). Unfortunately, the observed conformation of the C-terminal tail precluded direct interaction with the active-site disulfide͞dithiol group, suggesting an additional catalytically competent structural arrangement must exist.…”

“…The precise mechanism of selenenylsulfide͞disulfide exchange has not yet been determined for mammalian TrxRs, but studies of D. melanogaster (DmTrxR1) (26,48,49) and P. falciparum (27,37,50) high-M r TrxRs provide considerable insight. DmTrxR1 exhibits significant sequence homology to mammalian TrxRs but is not a selenoprotein (26,48).…”

Dithiol Proteins as Guardians of the Intracellular Redox Milieu in Parasites: Old and New Drug Targets in Trypanosomes and Malaria‐Causing Plasmodia

“…A key intermediate in catalysis is a charge transfer complex (the thiolate of Cys93 is the donor and flavin is the acceptor) characterized by a long-wavelength absorbance around 540 nm. [183,184] A high-throughput-screening program with 350 000 compounds of the Pfizer library yielded about 30 compounds that inhibit thioredoxin reductase. The bewildering chemical diversity among these hits was resolved by Davioud-Charvet et al, who noted that most of them were Mannich bases.…”

“…The gene was identified in the genome of P. falciparum, cloned, and expressed as a recombinant functional flavoenzyme. [152,[182][183][184] Subsequently, Müller and co-workers used gene-ablation and gene-substitution techniques to show that thioredoxin reductase is an essential enzyme for the blood stages of P. falciparum. [153,154] These studies validated thioredoxin reductase as a drug target.…”

Identification of Acid-Base Catalytic Residues of High-Mr Thioredoxin Reductase from Plasmodium falciparum

“…These homodimeric proteins contain three redox-active centers that are in redox communication: a tightly bound FAD, a pair of redox-active cysteines on one of the subunits that we will refer to as the N-terminal pair (Cys-88 and Cys-93 in Scheme 1, for PfTrxR), and a pair of redox-active cysteines or a cysteine-selenocysteine pair that we will refer to as the C-terminal pair (Cys-535Ј and Cys-540Ј, for PfTrxR) located on the second subunit (15,22,23,24). The reductive and oxidative half-reactions of Drosophila melanogaster TrxR (DmTrxR) have been examined in some detail to obtain a better understanding of the underlying catalytic mechanism of high-M r TrxRs.…”