Cloned (Comment) | Organism |
---|---|
gene glr0719, recombinant enzyme expression | Gloeobacter violaceus |
Crystallization (Comment) | Organism |
---|---|
purified transient complex between Fdx and GvDTR, 1.5fold molar excess of GvFdx1 over GvDTR, vapor diffusion method, mixing of protein in 10 mM Tris-HCl, pH 8.0, with an equal volume of mother liquor consisting of 40% v/v PEG 300, 100 mM sodium cacodylate-HCl, pH 6.5, and 200 mM sodium acetate, at 20°C, X-ray diffraction structure determination and analysis at 2.23-2.90 A resolution, molecular replacement using the crystal structures of GvDTR (PDB ID 5J60) and Fdx1 from Synechocystis (PDB ID 1OFF) as templates | Gloeobacter violaceus |
Protein Variants | Comment | Organism |
---|---|---|
additional information | deletion of the C-terminal tail in GvDTR abolishing enzyme activity | Gloeobacter violaceus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 reduced ferredoxin + thioredoxin disulfide | Gloeobacter violaceus | - |
2 oxidized ferredoxin + thioredoxin + 2 H+ | - |
? | |
2 reduced ferredoxin + thioredoxin disulfide | Gloeobacter violaceus ATCC 29082 | - |
2 oxidized ferredoxin + thioredoxin + 2 H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Gloeobacter violaceus | Q7NMP6 | - |
- |
Gloeobacter violaceus ATCC 29082 | Q7NMP6 | - |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
2 reduced ferredoxin + thioredoxin disulfide = 2 oxidized ferredoxin + thioredoxin + 2 H+ | reaction mechanism, overview | Gloeobacter violaceus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 reduced ferredoxin + thioredoxin disulfide | - |
Gloeobacter violaceus | 2 oxidized ferredoxin + thioredoxin + 2 H+ | - |
? | |
2 reduced ferredoxin + thioredoxin disulfide | - |
Gloeobacter violaceus ATCC 29082 | 2 oxidized ferredoxin + thioredoxin + 2 H+ | - |
? | |
additional information | reduced ferredoxin (Fdx) is the electron donor for the DTR enzyme from cyanobacterium Gloeobacter violaceus (GvDTR) | Gloeobacter violaceus | ? | - |
- |
|
additional information | reduced ferredoxin (Fdx) is the electron donor for the DTR enzyme from cyanobacterium Gloeobacter violaceus (GvDTR) | Gloeobacter violaceus ATCC 29082 | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
homodimer | GvDTR is a homodimer with each monomer composed of two conserved Rossmann-type modules that form the FAD-binding and redox-active disulfide domains | Gloeobacter violaceus |
Synonyms | Comment | Organism |
---|---|---|
Fdx flavin-thioredoxin reductase | - |
Gloeobacter violaceus |
Fdx-dependent thioredoxin reductase | - |
Gloeobacter violaceus |
ferredoxin-dependent thioredoxin reductase | - |
Gloeobacter violaceus |
FFTR | - |
Gloeobacter violaceus |
FTR | - |
Gloeobacter violaceus |
glr0719 | - |
Gloeobacter violaceus |
GvDTR | - |
Gloeobacter violaceus |
iron-sulfur ferredoxin-dependent thioredoxin reductase | - |
Gloeobacter violaceus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Gloeobacter violaceus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.6 | - |
assay at | Gloeobacter violaceus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | in each monomer, two conserved Rossmann-type modules form the FAD-binding. The pi-stacking interaction between the side chain of the conserved tryptophan at the C-terminal tail of a monomer and the isoalloxazine ring of the FAD of an adjacent monomer at its re-face. This interaction seems to protect the flavin from the solvent, a distinctive feature of the GvDTR enzyme not found in other flavin thioredoxin reductases. The C-terminal extension stabilizes the semiquinone state of the flavin | Gloeobacter violaceus | |
Ferredoxin | Fdx, iron-sulfur ferredoxin, enzyme-Fdx interaction analysis, overview. Investigation of GvDTR and GvFdx1 or GvFdx2 as functional redox partners by analyzing the reduction of photosynthetic m-type thioredoxin (Trxm) in a mixture containing NADPH, the redox pair Anabaena ferredoxin-NADP+ reductase (AnFNR)/GvFdx1 or AnFNR/GvFdx2, GvDTR, and Gloeobacter Trx-m (GvTrxm). The redox state of GvTrx-m is examined with the thiol-specific reagent 4-acetamido-40-maleimidyldistilbene-2,2'-disulfonic acid (AMS), separating the reduced and oxidized proteins with nonreducing SDS-PAG.Only GvFdx1 with the complete reactants significantly increases the reduced/oxidized ratio of GvTrx-m, prompting the conclusion that GvFFTR interacts with GvFdx1 but not with GvFdx2, which has a unique C-terminal extension. GvFdx1 is a functional electron-delivering partner for GvDTR. GvFdx1 binding to GvDTR is strictly dependent on the presence of the enzyme's C-terminal tail. GvFdx1 (petF, gvip492) and GvFdx2 (gvip440) are recombinantly expressed as N-terminally His8-tagged proteins, with a TEV cleavage site preceding the Fdx sequence, in Escherichia coli Rosetta (DE3) pLys cells | Gloeobacter violaceus |
General Information | Comment | Organism |
---|---|---|
evolution | some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack thioredoxin reductase flavoenzyme (NTR) and (Fdx)-dependent thioredoxin reductase (FTR) but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor is Fdx. This cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. The FFTR is spread within the cyanobacteria phylum. By substituting for FTR, it connects the reduction of target proteins to photosynthesis. FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments. Cyanobacterial Fdx-dependent thioredoxin reductases might have diverged early in the evolution into flavo- or metalloenzymes | Gloeobacter violaceus |
malfunction | deletion of the C-terminal tail does not significantly affect structure and both GvDTR and GvDTR_DELTAtail proteins are properly folded. The mutant enzyme is able to reduce Trx when a non-physiological electron donor (dithionite) is used | Gloeobacter violaceus |
additional information | crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. A unique feature of GvDTR is the presence of a C-terminal tail with a conserved aromatic amino acid that stacks onto the isoalloxazine ring of the FAD of the adjacent monomer. GvFdx1 binding to GvDTR is strictly dependent on the presence of the enzyme's C-terminal tail. Conformations adopted by GvDTR during its catalytic cycle, detailed overview | Gloeobacter violaceus |
physiological function | thioredoxin reductases control the redox state of thioredoxins (Trxs), ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. The FFTR/Trx system of Gloeobacter is able to reduce CP12, a small protein functional in the regulation of two enzymes of the Calvin-Benson cycle in oxygenic photosynthetic organisms-phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). FFTR can substitute for FTR in light-linked redox regulation in Gloeobacter | Gloeobacter violaceus |