Leopold Flohé

Diversity of glutathione peroxidases.

Publisher Summary This chapter focuses on the diversity of glutathione peroxidases. Selenium was identified as a toxic factor for grazing animals in the first half of the twentieth century and since then has been considered hazardous. Only long after the identification of the first selenoenzymes in bacteria and mammals was a Recommended Dietary Allowance gradually established. In fact, the putative biological roles of the selenoenzymes, particularly those of the glutathione peroxidases (GPX), proved instrumental in the understanding of selenium deficiency syndromes in livestock and humans, although the emerging complexity of selenium enzymology still precludes definitive conclusions. The selenium-dependent peroxidases have long been considered a late achievement of evolution, as they were only detected in vertebrates. This view now has to be revised. Whether the common ancester of the GPX superfamily was a selenoprotein or a cysteine-containing homolog cannot be deduced from the available sequences. The only prokaryotic member of the superfamily detected so far, a cobalamine-binding protein of Escherichia coli , does not contain selenocysteine, and despite ongoing efforts, functionally active glutathione peroxidases have not yet been found in prokaryotes.

The fairytale of the GSSG/GSH redox potential

BACKGROUNDnThe term GSSG/GSH redox potential is frequently used to explain redox regulation and other biological processes.nnnSCOPE OF REVIEWnThe relevance of the GSSG/GSH redox potential as driving force of biological processes is critically discussed. It is recalled that the concentration ratio of GSSG and GSH reflects little else than a steady state, which overwhelmingly results from fast enzymatic processes utilizing, degrading or regenerating GSH.nnnMAJOR CONCLUSIONSnA biological GSSG/GSH redox potential, as calculated by the Nernst equation, is a deduced electrochemical parameter based on direct measurements of GSH and GSSG that are often complicated by poorly substantiated assumptions. It is considered irrelevant to the steering of any biological process. GSH-utilizing enzymes depend on the concentration of GSH, not on [GSH](2), as is predicted by the Nernst equation, and are typically not affected by GSSG. Regulatory processes involving oxidants and GSH are considered to make use of mechanistic principles known for thiol peroxidases which catalyze the oxidation of hydroperoxides by GSH by means of an enzyme substitution mechanism involving only bimolecular reaction steps.nnnGENERAL SIGNIFICANCEnThe negligibly small rate constants of related spontaneous reactions as compared with enzyme-catalyzed ones underscore the superiority of kinetic parameters over electrochemical or thermodynamic ones for an in-depth understanding of GSH-dependent biological phenomena. At best, the GSSG/GSH potential might be useful as an analytical tool to disclose disturbances in redox metabolism. This article is part of a Special Issue entitled Cellular Functions of Glutathione.

CHANGING PARADIGMS IN THIOLOGY: FROM ANTIOXIDANT DEFENSE TOWARD REDOX REGULATION

The history of free radical biochemistry is briefly reviewed in respect to major trend shifts from the focus on radiation damage toward enzymology of radical production and removal and ultimately the role of radicals, hydroperoxides, and related fast reacting compounds in metabolic regulation. Selected aspects of the chemistry of radicals and hydroperoxides, the enzymology of peroxidases, and the biochemistry of adaptive responses and regulatory phenomena are compiled and discussed under the perspective of how the fragments of knowledge can be merged to biologically meaningful concepts of regulation. It is concluded that (i) not radicals but H(2)O(2), hydroperoxides, and peroxynitrite are the best candidates for oxidant signals, (ii) peroxidases of the GPx and Prx family or functionally equivalent proteins have the chance to specifically sense hydroperoxides and to transduce the oxidant signal, (iii) redox signaling proceeds via reactions known from thiol peroxidase and redoxin chemistry, (iv) proximal targets are proteins that are modified at SH groups, and (v) redoxins are documented signal transducers but also used as terminators. The importance of kinetics for forward signaling and for sensitivity modulation by competition is emphasized and ways to restore resting conditions are discussed. Research needs to validate emerging concepts are outlined.

The labour pains of biochemical selenology: the history of selenoprotein biosynthesis.

The serendipitous discoveries leading to the present knowledge on seleniums role in biology are reviewed. Detected in 1818 as by-product of sulphuric acid production, selenium first attracted medical attention as an industrial hazard. In parallel selenium intoxication was recognized as cause of life stock diseases. Reports on teratogenic effects and carcinogenicity of selenium followed since the middle of the past century. In 1954 first hints towards specific biological functions of selenium were contributed from microbiology, and its essentiality for mammalian life was discovered in 1957. Independent and unrelated studies led to the identification of selenium as an integral constituent of one mammalian and two bacterial enzymes in the early 70ies followed by the identification of selenocysteine in these proteins. In the 80ies, independent sequencing of selenoproteins and cloned DNAs revealed that the selenocysteine of selenoproteins is encoded by the termination codon TGA (UGA). Recoding of TGA as selenocysteine codon by secondary mRNA structures was first elucidated by molecular genetics in bacteria and later in mammals. During the 90ies, finally, the basic principles of selenoprotein synthesis were worked out by molecular biology tools. The article closes with spotlight comments on proven and potential biomedical benefits of selenium and related research deficits.

Selenoproteins of the glutathione system

The protein family of glutathione peroxidases (GPx) is found throughout the entire life kingdoms. Five distinct molecular clades characterized by an active site selenocysteine residue may coexist in vertebrates. All selenocysteine-containing GPx types reduce hydroperoxides with rate constants k’1 near 107 M−1 sec−1, while the cysteine homologs are poor peroxidases. The scope of accepted hydroperoxides increases from the cytosolic and gastrointestinal type to the extracellular type and phospholipid hydroperoxide GPx, while the specificity for GSH declines in this order. Compelling evidence defines cGPx as a device to detoxify H2O2 and soluble hydroperoxides. Being dispensable for survival, cGPx is nevertheless essential to maintain hydroperoxide homeostasis, as demonstrated by mimicking the development of Keshan disease in cGPx(−/−) mice. Being unable to substitute for cGPx in challenged cGPx(−/−) mice, the functions of other GPxs have to be sought in local regulation of peroxide-dependent processes, e. g., silencing leukotriene biosynthesis, dampening cytokine-dependent NFκB activation, regulating apoptosis and sperm differentiation by PHGPx. The functional divergence within the GPx family is further underscored by different mechanisms of transcriptional control.

The trypanothione system and the opportunities it offers to create drugs for the neglected kinetoplast diseases.

Parasitic trypanosomatids (Kinetoplastida) are the causative agents of devastating and hard-to-treat diseases such as African sleeping sickness, Chagas disease and various forms of Leishmaniasis. Altogether they affect > 30 Million patients, account for half a million fatalities p.a. and cause substantial economical problems in the Third World due to human morbidity and life stock losses. The design of efficacious and safe drugs is expected from inhibition of metabolic pathways that are unique and essential to the parasite and absent in the host. In this respect, the trypanothione system first detected in the insect-pathogenic trypanosomatid Crithidia fasciculata qualified as an attractive drug target area. The existence of the system in pathogenic relatives was established by homology cloning and PCR. The vital importance of the system was verified in Trypanosoma brucei by dsRNA technology or knock-out in other trypanosomatids, respectively, and is explained by its pivotal role in the parasites antioxidant defense and DNA synthesis. The key system component is the bis-glutathionyl derivative of spermidine, trypanothione. It is the proximal reductant of tryparedoxin which substitutes for thioredoxin-, glutaredoxin- and glutathione-dependent reactions. Heterologous expression, functional characterization and crystallization of recombinant system components finally enable structure-based rational inhibitor design.

Regulation of Glutathione Peroxidases

The glutathione peroxidases (GPx) belong to a superfamily of phylogenetically related proteins of diverse functions.1 The members of the superfamily containing a selenocysteine residue in their catalytic centers are highly efficient peroxidases reacting with a variety of hydroperoxides at rate constants of greater than 106 M-1 s-1.1 A cysteine residue in homologous position is catalytically less effective by about three orders of magnitude.2,3 Such GPx-like proteins, although still potential redox catalysts, can not be rated as peroxidases, if this term is to designate enzymes that must remove peroxides rapidly from a biological environment.

The trypanothione system and its implications in the therapy of trypanosomatid diseases

Biosynthesis and the use of trypanothione, a redox metabolite of parasitic trypanosomatids, are reviewed here with special emphasis on the development of trypanocidal drugs. This metabolic system is unique to and essential for the protozoal parasites. Selective inhibition of key elements of trypanothione metabolism, therefore, promises eradication of the parasites without affecting the host. Considering the metabolic importance and drugability of system components, inhibition of the enzymes for regeneration and de novo synthesis of trypanothione is rated as the most promising approach, while related peroxidases and redoxins are disregarded as targets because of limited chances to achieve selective inhibition. The organizational need to exploit the accumulating knowledge of trypanosomatid metabolism for medical practice is briefly addressed.

Selenium and male reproduction.

Selenium deficiency has long been documented to result in impaired male fertility of rats, mice and boars. The prominent feature of selenium-deficient spermatozoa is a distorted architecture of the mid piece, where normally the mitochondria are embedded into a keratinous matrix called the mitochondrial capsule. This material, which contains most of the selenium of sperm, is composed of oxidatively cross-linked proteins, a major component being the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx). PHGPx is abundantly synthesized in round spermatids under indirect control of testosterone. In late phase of spermatogenesis, the active soluble peroxidase is transformed into an enzymatically inactive structural protein by an oxidative process that is not understood in detail. Likely, it involves oligomerization of PHGPx itself, cross-linking of PHGPx with the sperm mitochondrion-associated cysteine-rich protein (SMCP) and other cysteine-rich proteins and selenadisulfide reshuffling with or without the aid of thioredoxin-glutathione reductase.

Molecular Dynamics Reveal Binding Mode of Glutathionylspermidine by Trypanothione Synthetase

The trypanothione synthetase (TryS) catalyses the two-step biosynthesis of trypanothione from spermidine and glutathione and is an attractive new drug target for the development of trypanocidal and antileishmanial drugs, especially since the structural information of TryS from Leishmania major has become available. Unfortunately, the TryS structure was solved without any of the substrates and lacks loop regions that are mechanistically important. This contribution describes docking and molecular dynamics simulations that led to further insights into trypanothione biosynthesis and, in particular, explains the binding modes of substrates for the second catalytic step. The structural model essentially confirm previously proposed binding sites for glutathione, ATP and two Mg2+ ions, which appear identical for both catalytic steps. The analysis of an unsolved loop region near the proposed spermidine binding site revealed a new pocket that was demonstrated to bind glutathionylspermidine in an inverted orientation. For the second step of trypanothione synthesis glutathionylspermidine is bound in a way that preferentially allows N1-glutathionylation of N8-glutathionylspermidine, classifying N8-glutathionylspermidine as the favoured substrate. By inhibitor docking, the binding site for N8-glutathionylspermidine was characterised as druggable.