thiI encodes a tRNA sulfurtransferase (EC 2.8.1.4) that catalyzes the ATP-dependent installation of 4-thiouridine (s4U) at position 8 of tRNA, with sulfur supplied by the cysteine desulfurase IscS via a persulfide intermediate. In Pseudomonas putida KT2440, deletion of thiI drastically reduces bulk-tRNA s4U (Fuchs et al. 2023, mass spectrometry), providing direct organism-specific evidence that ThiI is the s4U writer. The HAMAP-Rule annotation also assigns a second sulfur-transfer reaction to the ThiS sulfur-carrier protein, a step in thiazole/thiamine biosynthesis, though this thiamine role is currently an inference from family knowledge rather than KT2440-specific experimental proof.
Definition: Catalysis of sulfur transfer to the sulfur carrier protein ThiS to form ThiS-thiocarboxylate for thiazole biosynthesis.
Justification: ThiI has a second UniProt/Rhea-supported sulfur-transfer reaction to ThiS, but GOA only has broad sulfurtransferase activity plus thiazole/thiamine process annotations.
Parent term: sulfurtransferase activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000049 tRNA binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: tRNA binding is a genuine ThiI activity (the THUMP domain recognizes the tRNA 3'-CCA terminus) but is mechanistic context rather than the core catalytic function. Reason: ThiI acts on tRNA substrates, so binding is context rather than the core catalytic function. The falcon deep research confirms the THUMP RNA-binding domain captures the tRNA CCA end, consistent with the UniProt THUMP domain annotation (residues 63-167). Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt transfer of a sulfur to tRNA file:PSEPK/thiI/thiI-deep-research-falcon.md the **THUMP domain captures the tRNA 3β²-CCA terminus** |
| GO:0002937 tRNA 4-thiouridine biosynthesis | IEA GO_REF:0000118 | ACCEPT | Summary: tRNA 4-thiouridine biosynthesis is the core ThiI biological process and is now backed by direct organism-specific genetic evidence in P. putida KT2440, not merely IEA transfer. Reason: UniProt describes ATP-dependent sulfur transfer to tRNA to produce 4-thiouridine in position 8, and falcon deep research cites Fuchs et al. 2023 showing that a P. putida KT2440 deletion of thiI drastically reduces bulk-tRNA s4U (mass spectrometry), directly demonstrating that ThiI is the s4U writer in this organism. This is the central biological process for the gene. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt produce 4-thiouridine in position 8 of tRNAs file:PSEPK/thiI/thiI-deep-research-falcon.md deletion of **thiI** causes a **drastic reduction of s4U in bulk tRNA** file:PSEPK/thiI/thiI-deep-research-falcon.md a **ΞthiI** strain shows **drastically reduced** s4U relative to wild type |
| GO:0003723 RNA binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: RNA binding is a broad parent-style annotation for a tRNA-specific enzyme. Reason: The tRNA binding and tRNA sulfurtransferase annotations are more informative. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt transfer of a sulfur to tRNA |
| GO:0004810 CCA tRNA nucleotidyltransferase activity | IEA GO_REF:0000002 | REMOVE | Summary: CCA tRNA nucleotidyltransferase activity is inconsistent with ThiI biology. Reason: ThiI is a sulfurtransferase/tRNA 4-thiouridine synthase, not a CCA-adding tRNA nucleotidyltransferase; this InterPro-derived row appears to be a bad parent/family propagation. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt tRNA sulfurtransferase |
| GO:0005524 ATP binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: ATP binding is required for the sulfur-transfer chemistry (uridine adenylation) but is not the specific function. Reason: The core annotation should be tRNA-uracil-4 sulfurtransferase activity. ATP binding is the mechanistic prerequisite - falcon deep research describes ThiI as an ATP-dependent tRNA-modifying enzyme that activates the target uridine by adenylation, consistent with the four UniProt ATP-binding sites. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt ATP-dependent transfer of a sulfur to tRNA file:PSEPK/thiI/thiI-deep-research-falcon.md ATP-dependent** tRNA-modifying enzyme |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Cytoplasm is the expected localization for a bacterial soluble enzyme that acts on mature cellular tRNA. Reason: Location is useful context but not the defining function. Falcon deep research notes that no KT2440-specific localization experiment exists, but the substrate is cellular tRNA, implying a cytosolic/intracellular site of action consistent with the UniProt cytoplasm annotation. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm file:PSEPK/thiI/thiI-deep-research-falcon.md acting on mature cellular tRNA |
| GO:0005829 cytosol | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: Cytosol is expected localization but redundant with cytoplasm for this review. Reason: The TreeGrafter cytosol row can be retained as non-core context; falcon deep research places the site of action in the cytosol, acting on cellular tRNA. Supporting Evidence: file:PSEPK/thiI/thiI-deep-research-falcon.md The functional context is **intracellular/cytosolic**, acting on mature cellular tRNA. |
| GO:0009229 thiamine diphosphate biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: ThiI contributes to thiamine diphosphate biosynthesis through ThiS sulfur transfer, though the organism-specific evidence is weaker than for the tRNA s4U role. Reason: UniProt (HAMAP-Rule MF_00021) states that ThiI transfers sulfur to ThiS-thiocarboxylate as a step in thiazole/thiamine biosynthesis, with the second catalytic-activity reaction explicitly annotated. Falcon deep research notes that for KT2440 the thiamine-biosynthesis role remains an inference from ThiI-family knowledge rather than KT2440-specific proof, so this is retained as a well-supported family-level annotation rather than the gene's most directly demonstrated process. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt step in the synthesis of thiazole, in the thiamine biosynthesis pathway file:PSEPK/thiI/thiI-deep-research-falcon.md any thiamine-biosynthesis role remains an inference from family knowledge |
| GO:0016783 sulfurtransferase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: Sulfurtransferase activity is a broad parent of ThiI's specific tRNA-uracil-4 sulfurtransferase activity. Reason: The specific tRNA-uracil-4 sulfurtransferase activity (GO:0140741) should be preferred. Falcon deep research describes the canonical ThiI mechanism as an ATP-dependent adenylating sulfurtransferase, but the specific tRNA-acting term is more informative than the broad parent. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt tRNA sulfurtransferase file:PSEPK/thiI/thiI-deep-research-falcon.md adenylation of the target uridine and sulfur transfer via persulfide chemistry |
| GO:0034227 tRNA thio-modification | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: tRNA thio-modification is correct but less specific than tRNA 4-thiouridine biosynthesis. Reason: GO:0002937 captures the product and position of ThiI's direct tRNA modification. Falcon deep research confirms ThiI installs 4-thiouridine (s4U) in cellular tRNA, the specific thio-modification, so the broad parent thio-modification term is redundant with the more specific s4U biosynthesis term. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt produce 4-thiouridine in position 8 of tRNAs file:PSEPK/thiI/thiI-deep-research-falcon.md responsible for installing **4-thiouridine (s4U)** in cellular tRNA |
| GO:0052837 thiazole biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Thiazole biosynthesis is supported by ThiI sulfur transfer to the ThiS sulfur-carrier protein. Reason: UniProt (HAMAP-Rule MF_00021) explicitly links the ThiS sulfur-carrier reaction to thiazole synthesis in thiamine biosynthesis. Falcon deep research corroborates that ThiI links to thiamine (thiazole) biosynthesis at the family level, while noting this role is a ThiI-family inference rather than a KT2440-specific measurement. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt transfer of sulfur to the sulfur carrier protein ThiS file:PSEPK/thiI/thiI-deep-research-falcon.md links to **thiamine (thiazole) biosynthesis** |
| GO:0140741 tRNA-uracil-4 sulfurtransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: tRNA-uracil-4 sulfurtransferase activity is the specific core molecular function, supported by EC 2.8.1.4/Rhea and by genetic evidence that thiI is required for tRNA s4U in KT2440. Reason: The EC/Rhea-supported UniProt entry describes the ATP-dependent sulfur transfer to tRNA uridine. Falcon deep research adds the mechanistic model (ATP-dependent adenylation of the target uridine followed by sulfur transfer via a persulfide supplied by the cysteine desulfurase IscS) and the KT2440-specific loss-of-function evidence, making this the best-supported molecular function. Supporting Evidence: file:PSEPK/thiI/thiI-uniprot.txt ATP-dependent transfer of a sulfur to tRNA file:PSEPK/thiI/thiI-deep-research-falcon.md ATP-dependent thiolation of uridine in tRNA to form 4-thiouridine (s4U), typically at position 8 file:PSEPK/thiI/thiI-deep-research-falcon.md sulfur is mobilized from **L-cysteine by the cysteine desulfurase IscS** |
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Download this section (compressed HTML)Q: How is ThiI flux partitioned between tRNA 4-thiouridine synthesis and ThiS sulfur transfer in KT2440?
Q: Which cysteine desulfurase (IscS-like) is the in vivo sulfur donor for ThiI in P. putida KT2440? The IscS-persulfide model is inferred from E. coli by homology but has not been biochemically reconstituted in this organism.
Experiment: Quantify tRNA s4U and thiamine/thiazole pathway intermediates in thiI mutants that separate tRNA-binding and ThiS-transfer surfaces.
Type: separation-of-function mutant analysis
Experiment: In vitro reconstitution of the KT2440 ThiI sulfur relay with candidate cysteine desulfurases to identify the physiological persulfide donor and confirm the active-site cysteine (homologous to E. coli Cys456) used for sulfur transfer.
Type: biochemical reconstitution
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