Functional metagenomics of the thioredoxin superfamily.

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

Phylogenetic distribution and structural analyses of cyanobacterial glutaredoxins (Grxs).

Glutaredoxins (Grxs), the oxidoreductase proteins, are involved in several cellular processes, including maintenance of cellular redox potential and iron-sulfur homeostasis. The analysis of 503 amino acid sequences from 167 cyanobacterial species led to the identification of four classes of cyanobacterial Grxs, i.e., class I, II, V, and VI Grxs. Class III and IV Grxs were absent in cyanobacteria. Class I and II Grxs are single module oxidoreductase while class V and VI Grxs are multimodular proteins having additional modules at their C-terminal and N-terminal end, respectively. Furthermore, class VI Grxs were exclusively present in marine cyanobacteria. We also report the identification of class VI Grxs with two novel active site motif compositions. Detailed phylogenetic analysis of all four classes of Grxs revealed the presence of several subgroups within each class of Grx having variable dithiol and/or monothiol catalytic active site motif and putative glutathione binding sites. However, class II Grxs possess CGFS-type highly conserved monothiol catalytic active site motif. Sequence analysis confirmed the highly diverse nature of Grx proteins in terms of their amino acid composition; though, sequence diversity does not affect the overall 3D structure of cyanobacterial Grxs. The active site residues and putative GSH binding residues are uncharged amino acids which are present on the surface of the protein. Additionally, the presence of hydrophilic residues at the surface of Grxs confirms their solubility. Protein-ligand interaction analysis identified novel glutathione binding sites on Grxs. Regulation of Grxs encoding genes expression by light quality and quantity as well as salinity suggests their role in determining the fitness of organisms under abiotic factors.

The biological roles of glutaredoxins.

Grxs (glutaredoxins) are small ubiquitous redox enzymes. They are generally involved in the reduction of oxidative modifications using glutathione. Grxs are not only able to reduce protein disulfides and the low-molecular-mass antioxidant dehydroascorbate, but also represent the major enzyme class responsible for deglutathionylation reactions. Functional proteomics, including interaction studies, comparative activity measurements using heterologous proteins and structural analysis are combined to provide important insights into the crucial function of Grxs in cellular redox networks. Summarizing the current understanding of Grxs, with a special focus on organelle-localized members across species, genus and kingdom boundaries (including cyanobacteria, plants, bacteria, yeast and humans) lead to two different classifications, one according to sequence structure that gives insights into the diversification of Grxs, and another according to function within the cell that provides a basis for assessing the different roles of Grxs.

Evolution and diversity of glutaredoxins in photosynthetic organisms.

The genome sequencing of prokaryotic and eukaryotic photosynthetic organisms enables a comparative genomic study of the glutaredoxin (Grx) family. The analysis of 58 genomes, using a specific motif composed of the active site sequence and of amino acids involved in glutathione binding, led to an updated classification of Grxs into six classes. Only two classes (I and II) are common to all photosynthetic organisms. Eukaryotes and cyanobacteria have two specific Grx classes (classes III and IV and classes V and VI, respectively). The classes IV, V and VI have not yet been identified and contain multimodular Grx fusions. In addition, putative Grx partners were identified from the presence of fusion proteins, the conservation of gene order in bacterial operons, and the gene co-occurrence. The genes encoding class II Grxs and BolA/YrbA proteins are frequently adjacent, in the same transcriptional orientation in prokaryote genomes and present in the same organisms.

Glutaredoxin systems.

Glutaredoxins utilize the reducing power of glutathione to maintain and regulate the cellular redox state and redox-dependent signaling pathways, for instance, by catalyzing reversible protein S-glutathionylation. Due to the general importance of these processes, glutaredoxins have been implied in various physiological and disease-related conditions, such as immune defense, cardiac hypertrophy, hypoxia-reoxygenation insult, neurodegeneration and cancer development, progression as well as treatment. The past years have seen an impressive gain of knowledge regarding new glutaredoxin systems and functions. This is true both with respect to new functions in redox regulation and also with respect to unexpected new ties to iron metabolism and iron-sulfur cluster biosynthesis. The aim of this review is to provide a state-of-the-art overview over these recent discoveries with a focus on aspects related to human health.

CGFS-type monothiol glutaredoxins from the cyanobacterium Synechocystis PCC6803 and other evolutionary distant model organisms possess a glutathione-ligated [2Fe-2S] cluster.

When produced in Escherichia coli, the CGFS-type monothiol Grxs from this organism (EcGrx4p) and the model cyanobacterium Synechocystis (SyGrx3p) exist as a dimeric iron-sulfur containing holoprotein or as a monomeric apoprotein in solution. Spectroscopic and site-directed mutagenesis analyses show that the SyGrx3 holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteine located in the CGFS motif of each monomer and the cysteines of two molecules of glutathione. The biochemical characterization of several monothiol Grxs from the cyanobacteria Gloeobacter violaceus (GvGrx3p) and Thermosynechococcus elongatus (TeGrx3p), the yeast Saccharomyces cerevisiae (ScGrx3p, ScGrx4p, and ScGrx5p), the plant Arabidopsis thaliana (AtGrx5p), and human (HsGrx5p) indicate that the incorporation of a GSH-ligated [2Fe-2S] center is a common feature of prokaryotic and eukaryotic CGFS-active site monothiol Grxs. In light of these results, the involvement of these enzymes in the sensing of iron and/or the biogenesis and transfer of Fe-S cluster is discussed.