TYW1 (tRNA-yW synthesizing protein 1) is a cytoplasmic radical S-adenosyl-L-methionine (SAM) flavoenzyme. It catalyses the second step of wybutosine (yW) biosynthesis, the hypermodification of guanosine 37 next to the anticodon of tRNA(Phe). Starting from N1-methylguanosine (m1G37), made by TRMT5, TYW1 condenses carbons 2 and 3 of pyruvate onto the base to form the tricyclic 4-demethylwyosine (imG-14), releasing carbon 1 of pyruvate as CO2. The enzyme has an N-terminal flavodoxin-like domain that binds FMN and supplies reducing equivalents from NAD(P)H, and a C-terminal radical SAM domain. The radical SAM domain carries two [4Fe-4S] clusters: one bound by the CX3CX2C motif that cleaves SAM to a 5'-deoxyadenosyl radical, and an auxiliary cluster that binds pyruvate. imG-14 is then elaborated by TYW2/TRMT12, TYW3, LCMT2/TYW4 and, in human, TYW5 to (hydroxy)wybutosine, which stabilises codon-anticodon pairing and keeps the reading frame at Phe codons. Most of the enzymology was established for the yeast and archaeal orthologues. In human cells, TYW1 knockout abolishes hydroxywybutosine and lowers UUU-codon translation. Biallelic loss-of-function variants cause primary microcephaly with intellectual disability and cerebral palsy. Human chromosome 7 also carries a closely related paralogue, TYW1B.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM domain (IPR007197) to the root catalytic activity term. TYW1 has a known specific reaction, tRNA 4-demethylwyosine synthase (EC 4.1.3.44), which the gene already carries as GO:0102521. Reason: The term is correct but is the root of the MF catalytic branch. The project rule for generic radical SAM rows is to MODIFY GO:0003824 to the specific reaction MF the gene carries. Here that is GO:0102521 tRNA-4-demethylwyosine synthase activity, an is_a descendant (via oxo-acid-lyase activity) already annotated to TYW1. The reaction is inferred from yeast Tyw1 and the archaeal enzymes. In human cells, loss of TYW1 abolishes the downstream hydroxywybutosine product (PMID:38706838). Proposed replacements: tRNA-4-demethylwyosine synthase activity Supporting Evidence: file:human/TYW1/TYW1-uniprot.txt Catalyzes the condensation of N-methylguanine with 2 carbon atoms from pyruvate to form the tricyclic 4-demethylwyosine, an intermediate in wybutosine biosynthesis. PMID:38706838 OHyW was completely undetectable in TYW1-knockout hESCs |
| GO:0008033 tRNA processing | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the tRNA wybutosine-synthesis family signature (IPR034556) to tRNA processing. TYW1 modifies tRNA(Phe) at position 37 as part of wybutosine synthesis, which falls under tRNA modification, itself a kind of tRNA processing. Reason: The term is correct but general. GO:0031591 wybutosine biosynthetic process is an is_a descendant of GO:0008033 (via GO:0006400 tRNA modification). TYW1 already carries it by IBA, and it is the precise process TYW1 performs a step of: the tricyclic ring-forming second step (PMID:17727881, PMID:16642040). Proposed replacements: wybutosine biosynthetic process Supporting Evidence: PMID:17727881 TYW1 catalyzes the second step to form the tricyclic ring structure, by cyclizing N(1)-methylguanosine. |
| GO:0010181 FMN binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the flavodoxin-like domain signatures (IPR001094, IPR008254). Eukaryotic TYW1 proteins carry an N-terminal flavodoxin domain that the archaeal homologues lack. Recombinant yeast Tyw1 co-purifies with FMN and is active with NAD(P)H alone, so the flavodoxin domain is the built-in electron donor that reduces the radical SAM cluster. Reason: Human TYW1 keeps the flavodoxin domain, including the phosphate-binding loop at residues 85-89 annotated as an FMN-binding feature in UniProt. FMN binding has been shown biochemically for the yeast orthologue (PMID:34184886). The term is as specific as GO allows, and the cofactor is part of the enzyme's catalytic mechanism, so the row is core. Supporting Evidence: PMID:34184886 eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues PMID:34184886 ScTYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone. file:human/TYW1/TYW1-uniprot.txt /ligand="FMN" |
| GO:0031591 wybutosine biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN000358104, grounded in yeast Tyw1 (SGD:S000006128), which is required for yW synthesis and catalyses the tricyclic ring-forming step. Human TYW1 directly does a step of the process: it forms imG-14 from m1G37-tRNA(Phe). Human TYW1-knockout hESCs lack hydroxywybutosine entirely. Reason: TYW1 performs a catalytic step of wybutosine synthesis, so it passes the participation test. The IBA node placement is supported by human data: TYW1 knockout in hESCs removes OHyW, and patient missense variants destabilise the protein and lower OHyW (PMID:38706838). This is the core process of the gene. Supporting Evidence: PMID:16642040 TYW1 is an iron-sulfur (Fe-S) cluster protein responsible for the tricyclic formation. PMID:38706838 OHyW was completely undetectable in TYW1-knockout hESCs PMID:38706838 these results demonstrated that TYW1 variant or depletion directly impeded the final formation of OHyW modification |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM domain. TYW1 binds iron-sulfur clusters specifically as [4Fe-4S] clusters: the radical SAM cluster and an auxiliary cluster. Reason: Project rule for generic radical SAM rows: MODIFY GO:0051536 to its descendant GO:0051539 4 iron, 4 sulfur cluster binding, which the gene already carries. Both TYW1 clusters are [4Fe-4S] (PMID:23043105, PMID:17727881). Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:23043105 these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the TYW1 family signature. TYW1 enzymes bind two [4Fe-4S] clusters. The radical SAM cluster is held by the CX3CX2C motif (human C416, C420, C423). The auxiliary cluster is held by a second, upstream cysteine motif (human C381, C394, C407, aligned to the ligands of the Pyrococcus abyssi enzyme), and it binds the pyruvate cosubstrate. Reason: Project rule: GO:0051539 is ACCEPT and core for active radical SAM enzymes. The radical SAM cluster is the catalytic cofactor. Both cluster ligand sets are conserved in human TYW1 (file:human/TYW1B/TYW1B-bioinformatics/RESULTS.md). Two [4Fe-4S] clusters have been shown in archaeal TYW1 (PMID:23043105) and modelled with supporting yeast mutagenesis (PMID:17727881). Supporting Evidence: PMID:17727881 Mutational analyses in yeast supported the validity of this complex model structure, which provides a structural insight into the radical reaction involving two [4Fe-4S] clusters to create a complex tricyclic base. PMID:23043105 these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster file:human/TYW1/TYW1-uniprot.txt /ligand_note="4Fe-4S-S-AdoMet" file:human/TYW1B/TYW1B-bioinformatics/RESULTS.md align to TYW1 C381/C394/C407, all three conserved in yeast Tyw1. |
| GO:0102521 tRNA-4-demethylwyosine synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA from the EC 4.1.3.44 / RHEA:36347 assignment: m1G37 in tRNA(Phe) + pyruvate + SAM = 4-demethylwyosine37 in tRNA(Phe) + 5'-deoxyadenosine + L-methionine + CO2 + H2O. This is the defining activity of the TYW1 family. It has been shown in vitro for archaeal and yeast TYW1 (pyruvate as the C2 donor, CO2 from C1), and TYW1 is required for wybutosine in human cells. Reason: This is the core molecular function. Human TYW1 conserves every catalytic element: the radical SAM triad, the auxiliary-cluster cysteines and the flavodoxin domain. The reaction stoichiometry is established in orthologues (PMID:22026549, PMID:36326713, PMID:34184886). Human TYW1 knockout abolishes the downstream OHyW (PMID:38706838). Direct in vitro assay of the human enzyme has not been reported, but the inference is strong. Supporting Evidence: PMID:22026549 Of the potential substrates examined, only incubation with pyruvate resulted in formation of 4-demethylwyosine. PMID:36326713 under conditions that preferentially transform CO2 and not formate to OAA file:human/TYW1/TYW1-uniprot.txt Catalyzes the condensation of N-methylguanine with 2 carbon atoms from pyruvate to form the tricyclic 4-demethylwyosine PMID:38706838 OHyW was completely undetectable in TYW1-knockout hESCs |
| GO:0005737 cytoplasm | IDA PMID:38706838 Wybutosine hypomodification of tRNAphe activates HERVK and i... | NEW | Summary: Immunostaining of wild-type TYW1 (and two patient variants) in HeLa cells shows cytoplasmic, not nuclear, localisation. This fits the cytoplasmic stage of wybutosine formation on tRNA(Phe) and TYW1's identification as a client of the cytosolic iron-sulfur assembly (CIA) targeting complex. Reason: GOA has no cellular component annotation for TYW1. Direct human imaging places it in the cytoplasm (PMID:38706838). This is kept at the cytoplasm level: the cytoplasm-marker channel was an ER antibody, and yeast Tyw1 is ER-localised, so association with the ER membrane is possible but has not been shown for human TYW1. Supporting Evidence: PMID:38706838 both wild-type (WT) and two variants of TYW1 showed clear localization in cytoplasm but not nucleus PMID:28178521 4-demethylwyosine synthase (TYW1) |
| GO:1904047 S-adenosyl-L-methionine binding | ISS PMID:36326713 Insertion of 4-Demethylwyosine in tRNA(Phe) Catalyzed by the... | NEW | Summary: TYW1 binds SAM at the unique iron of its radical SAM [4Fe-4S] cluster and cleaves it reductively on every turnover to generate the 5'-deoxyadenosyl radical. Reason: GOA has no SAM-binding row for TYW1, although the RADICAL_SAM project treats GO:1904047 as a core cosubstrate-binding function of every active radical SAM enzyme. As a molecular function, the participation test is met directly: TYW1 itself binds and cleaves SAM. The curated family rule records SAM as an exchangeable ligand of the radical SAM cluster, and TYW1 enzymology shows SAM cleavage on each turnover (PMID:36326713). Comparator check, against GOA rather than against this project's own proposals: GOA does annotate GO:1904047 to radical SAM enzymes in exactly this role, by UniRule -- ECOLI/queE (UR000100349), ECOLI/moaA and PSEPK/moaA (UR000101305), METEA/pqqE and PSEPK/pqqE (UR000098303). So the absence here is patchy UniRule coverage, which rules happen to carry the term, not a GO convention against annotating a radical SAM enzyme's own cosubstrate. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. Supporting Evidence: file:human/TYW1/TYW1-uniprot.txt The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:36326713 TYW1 requires reductive cleavage of SAM on each turnover. |
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Download this section (compressed HTML)Q: Has the 4-demethylwyosine synthase activity of human TYW1 been reconstituted in vitro, and does it require the flavodoxin domain or accept an external reductant?
Suggested experts: Bandarian V
Q: Did the TYW1 CRISPR knockout in hESCs also target the 95.7%-identical paralogue TYW1B? If not, does the complete loss of OHyW show that TYW1B cannot make up for TYW1 loss?
Q: Is human TYW1 attached to the ER membrane through its N-terminal hydrophobic segment, as yeast Tyw1 is localised to the ER?
Experiment: Express and anaerobically reconstitute human TYW1 (wild type and C381A/C394A/C407A auxiliary-cluster or C416A radical-SAM mutants). Assay imG-14 formation on m1G37 tRNA(Phe) by LC-MS, and track 5'-dA and CO2 from [1-13C]pyruvate.
Hypothesis: Recombinant human TYW1 converts m1G37-tRNA(Phe) and pyruvate into imG-14 in an NAD(P)H/FMN-dependent radical SAM reaction.
Type: in vitro enzymology
Experiment: Image endogenously tagged TYW1 in human cells with ER and cytosol markers, and use carbonate extraction and protease protection to test membrane integration of the N-terminal segment.
Hypothesis: Human TYW1 is anchored at the cytoplasmic face of the ER membrane.
Type: cell biology
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