Trypanothione reductase (TryR/TRYR) is the NADPH-dependent flavoprotein disulfide reductase that keeps the kinetoplastid-specific low-molecular-weight thiol trypanothione [N1,N8-bis(glutathionyl)spermidine] in its reduced dithiol form, T(SH)2. It is a homodimeric member of the class-I pyridine nucleotide-disulfide oxidoreductase family (the glutathione reductase/lipoamide dehydrogenase/mercuric reductase superfamily), with one FAD per subunit and a redox-active Cys52-Cys57 disulfide that transfers electrons from NADPH via FAD to trypanothione disulfide. The T. brucei enzyme strongly prefers NADPH over NADH and trypanothione disulfide over glutathione disulfide, so it does the job that glutathione reductase does in other eukaryotes. TryR lacks a mitochondrial presequence or a glycosomal PTS1 and is thought to act mainly in the cytosol. Reduced trypanothione supplies electrons to the tryparedoxin/peroxiredoxin systems that detoxify hydroperoxides and maintain thiol redox balance. TryR is essential: bloodstream trypanosomes with less than 10% of wild-type activity cannot grow, are highly sensitive to hydrogen peroxide, and fail to establish or sustain infection in mice. Because the trypanothione system has no counterpart in the mammalian host, TryR is a validated drug target.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004362 glutathione-disulfide reductase (NADPH) activity | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter transfer of glutathione reductase activity from the PTHR42737 (glutathione reductase) family node. Purified T. brucei TryR is highly specific for trypanothione disulfide and reduces glutathione disulfide only negligibly. Reason: Paralog/family-level over-annotation. Trypanothione reductase and glutathione reductase are sister enzymes with mutually exclusive disulfide substrate specificity, and the recombinant T. brucei enzyme shows a striking preference for T(S)2 over GSSG (PMID:19747949). The correct activity, trypanothione-disulfide reductase (NADPH) activity (GO:0015042), is already present as a separate annotation. Kinetoplastids have no glutathione reductase; trypanothione and TryR replace the GSH/GR system (PMID:10672177). Supporting Evidence: PMID:19747949 the kinetic and physical properties of TbTryR are consistent with trypanothione reductases from other trypanosomatids such as T. cruzi and L. donovani, especially the striking preference for T(S)2 over glutathione disulphide. PMID:19747949 However, the enzymes trypanothione reductase and glutathione reductase are highly specific for their respective disulphide substrates PMID:10672177 In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | MODIFY | Summary: UniProt SubCell mapping (Cytoplasm). Correct but less specific than cytosol, which the gene already carries. Reason: TryR is a soluble enzyme with no mitochondrial presequence (the sequence starts directly with the FAD-binding GxGxxG motif) and no canonical C-terminal PTS1, so it is expected to be cytosolic rather than glycosomal or mitochondrial. The more specific term cytosol (GO:0005829), already annotated, is preferred. Proposed replacements: cytosol Supporting Evidence: file:TRYBB/TPR/TPR-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. |
| GO:0005739 mitochondrion | IEA GO_REF:0000118 | UNDECIDED | Summary: TreeGrafter transfer from the glutathione reductase family node, where some members (e.g. dual-targeted fungal and animal GRs) have mitochondrial isoforms. Reason: T. brucei TryR has no recognizable N-terminal mitochondrial targeting sequence, and UniProt records only Cytoplasm. However, the subcellular distribution of TryR in bloodstream and procyclic T. brucei was studied directly by Smith, Opperdoes and Fairlamb (PMID:1779986), and the cached record has no abstract or full text, so the fractionation result could not be checked. The kinetoplastid mitochondrion does use trypanothione-dependent peroxidase systems, so a minor mitochondrial pool cannot be excluded. Left undecided pending access to that paper. |
| GO:0005829 cytosol | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter cytosol annotation. Consistent with the UniProt Cytoplasm assignment and with the soluble, untargeted nature of TryR. Reason: TryR maintains the intracellular reduced trypanothione pool, has no organellar targeting signal, and UniProt places it in the cytoplasm. Cytosol is the appropriate specific location. Supporting Evidence: file:TRYBB/TPR/TPR-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. PMID:1444271 Together with the NADPH-dependent flavoprotein, trypanothione reductase, the dithiol form of trypanothione provides an intracellular reducing environment in these parasites, substituting for glutathione and glutathione reductase found in the mammalian host. |
| GO:0006749 glutathione metabolic process | IEA GO_REF:0000118 | MODIFY | Summary: TreeGrafter transfer from glutathione reductases. TryR acts on trypanothione, a glutathione-spermidine conjugate, not on glutathione itself. Reason: The process TryR takes part in is trypanothione metabolism. It reduces trypanothione disulfide and has negligible activity on glutathione disulfide (PMID:19747949). GO:0046206 trypanothione metabolic process (verified in OLS) is the lineage-specific term that fits the enzyme's actual substrate. Proposed replacements: trypanothione metabolic process Supporting Evidence: PMID:19747949 TbTryR was highly specific for the electron donor (NADPH) and electron acceptor (T(S)2) in the 412 nm assay. PMID:1444271 Trypanosomatids differ from all other organisms in their ability to conjugate the sulfur-containing tripeptide, glutathione, and the polyamine, spermidine, to form trypanothione [N1,N8-bis(glutathionyl)spermidine]. |
| GO:0015036 disulfide oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO from the trypanothione reductase family signature (IPR001864). Correct but general. Reason: GO:0015042 trypanothione-disulfide reductase (NADPH) activity is an is_a descendant of this term (checked in OLS), is supported by kinetic data on the T. brucei enzyme, and is already annotated to this gene. Proposed replacements: trypanothione-disulfide reductase (NADPH) activity Supporting Evidence: PMID:19747949 TbTryR was highly specific for the electron donor (NADPH) and electron acceptor (T(S)2) in the 412 nm assay. |
| GO:0015042 trypanothione-disulfide reductase (NADPH) activity | IEA GO_REF:0000120 | ACCEPT | Summary: EC 1.8.1.12 / RHEA:16757 mapping. This is the defining catalytic activity of TryR, shown directly for recombinant T. brucei enzyme (NADPH-dependent reduction of trypanothione disulfide, Km for NADPH 0.77 uM). Reason: Core molecular function, supported by enzymology and structure of the T. brucei protein (PMID:19747949) and by the in vivo requirement for TryR activity (PMID:10672177). Supporting Evidence: PMID:19747949 TbTryR was highly specific for the electron donor (NADPH) and electron acceptor (T(S)2) in the 412 nm assay. PMID:19747949 Protection of the parasite against oxidative stress is achieved through the oxidation of the dithiol form of trypanothione (T(SH)2) into the disulphide form (T(S)2), followed by regeneration of T(SH)2 by the NADPH-dependent enzyme trypanothione reductase (TryR) |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO from generic pyridine nucleotide-disulfide oxidoreductase domains. True but uninformative. Reason: Replace with the specific supported activity GO:0015042, an is_a descendant already annotated to this gene. Proposed replacements: trypanothione-disulfide reductase (NADPH) activity |
| GO:0016668 oxidoreductase activity, acting on a sulfur group of donors, NAD(P) as acceptor | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO from the class-I pyridine nucleotide-disulfide oxidoreductase active-site signature. Correct but general. Reason: GO:0015042 is a direct descendant (verified in OLS) and is the experimentally supported activity for TryR. Proposed replacements: trypanothione-disulfide reductase (NADPH) activity |
| GO:0034599 cellular response to oxidative stress | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter transfer. Supported in T. brucei by TryR knockdown, which makes cells about 60-fold more sensitive to hydrogen peroxide. Reason: TryR does part of the work of the antioxidant response. It regenerates T(SH)2, the electron donor for tryparedoxin-dependent peroxide reduction, and TryR-depleted parasites are hypersensitive to H2O2 (PMID:10672177). Trypanothione and its enzymes defend against oxidant damage (PMID:1444271). Supporting Evidence: PMID:10672177 hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM) PMID:1444271 Trypanothione and its related enzymes are involved in defense against damage by oxidants, certain heavy metals, and possibly xenobiotics. |
| GO:0045454 cell redox homeostasis | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (InterPro + TreeGrafter). TryR keeps the trypanothione pool reduced and so maintains the intracellular thiol-reducing environment. Reason: Core biological role. Trypanothione and TryR provide the intracellular reducing environment in kinetoplastids, in place of the GSH/GR system (PMID:10672177, PMID:1444271). Supporting Evidence: PMID:10672177 In Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione-glutathione reductase system found in most other organisms. |
| GO:0050660 flavin adenine dinucleotide binding | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: FAD is a tightly bound prosthetic group, one per subunit, seen in the T. brucei crystal structure (PDB 2WBA). Reason: Correct and as specific as warranted. FAD binding is a cofactor property underlying the catalytic activity rather than the function itself. Supporting Evidence: PMID:19747949 TbTryR is a homodimer and each subunit of the final model comprises 489 amino acid residues, 1 FAD, 1 NADPH, 4 glycerol and 1 sulphate molecules. |
| GO:0098869 cellular oxidant detoxification | IEA GO_REF:0000108 | KEEP AS NON CORE | Summary: Inferred by inter-ontology link from glutathione-disulfide reductase activity, which is itself an incorrect annotation for TryR. The process is nonetheless plausible biologically, because TryR supplies reducing equivalents to the tryparedoxin/peroxiredoxin hydroperoxide-detoxification pathway. Reason: The inference source (GO:0004362) is wrong, but the claim holds independently. TryR-depleted trypanosomes cannot cope with H2O2 (PMID:10672177). TryR does not reduce peroxides itself; it regenerates the electron donor, so this is treated as a downstream, non-core role, with cell redox homeostasis as the core process. Supporting Evidence: PMID:10672177 hypersensitivity towards hydrogen peroxide (EC50 = 3.5 microM) was observed compared with the wild type (EC50 = 223 microM) |
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Download this section (compressed HTML)Q: Is any T. brucei TryR imported into the single mitochondrion, for example by an internal or alternative-start targeting signal, or is mitochondrial trypanothione reduced in the cytosol and imported? The 1991 fractionation study (PMID:1779986) should be checked against TrypTag localisation data for Tb927.10.10390.
Q: GO:0004362 glutathione-disulfide reductase activity is propagated by TreeGrafter from PANTHER PTHR42737 to kinetoplastid trypanothione reductases. Should the TryR subfamily (PTHR42737:SF2 / TIGR01423) be excluded from that node, given the experimentally established mutually exclusive substrate specificity?
Experiment: Endogenous-locus tagging with C- and N-terminal mNeonGreen, combined with digitonin titration and immunoblotting of cytosolic versus mitochondrial fractions in bloodstream and procyclic forms, using compartment markers.
Hypothesis: A fraction of TryR is located in the T. brucei mitochondrion and is needed to reduce trypanothione for mitochondrial tryparedoxin peroxidase.
Type: subcellular localization
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