Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent oxidoreductase that catalyzes the first committed step in ommochrome pigment biosynthesis in D. pealeii, converting L-tryptophan to N-formylkynurenine via oxidative cleavage of the indole ring. TDO is expressed in the retina and chromatophore layer of the skin, the two principal ommochrome-pigmented tissues. Crawford et al. (2020) demonstrated that CRISPR-Cas9 knockout of TDO in D. pealeii embryos efficiently eliminated pigmentation in both eyes and chromatophores with >90% disruption efficiency, while ink sac melanin pigmentation was unaffected. This was the first gene knockout achieved in any cephalopod. TDO inhibition with 680C91 also completely blocked pigmentation, confirming the enzyme is required for ommochrome synthesis. Notably, D. pealeii lacks an IDO (indoleamine 2,3-dioxygenase) ortholog, unlike Euprymna berryi where IDO can compensate for TDO loss. TDO belongs to the PANTHER family PTHR10138 (Tryptophan 2,3-dioxygenase), is predicted to function as a homotetramer (dimer of dimers), and contains a single heme cofactor per subunit. The gene spans 13 exons over 120 kb of genomic DNA.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004833 L-tryptophan 2,3-dioxygenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: L-tryptophan 2,3-dioxygenase activity (EC 1.13.11.11) is the defining enzymatic function of TDO. This IEA annotation is based on combined automated methods (InterPro, ARBA, PANTHER, UniRule). In D. pealeii, Crawford et al. (2020) demonstrated that CRISPR-Cas9 knockout of TDO eliminated ommochrome pigmentation, and pharmacological inhibition with 680C91 (a TDO-selective inhibitor) blocked pigmentation in both eyes and chromatophores [PMID:32735817]. The UniProt entry confirms TDO catalyzes L-tryptophan + O2 = N-formylkynurenine (Rhea:RHEA:24536). Phylogenetic analysis confirmed this gene as a bona fide TDO (distinct from IDO) [PMID:32735817]. Reason: This is the core enzymatic function of TDO, supported by sequence homology, PANTHER family assignment (PTHR10138), HAMAP rule MF_03020, and functional knockout data in this species showing loss of ommochrome pigmentation. Supporting Evidence: PMID:32735817 In invertebrates, Tryptophan, 2,3 Dioxygenase (TDO) catalyzes the first committed step in ommochrome biosynthesis, converting Tryptophan to N-Formylkyneurenine. PMID:32735817 A phylogenetic comparison of this sequence with diverse TDOs, and indolamine-2,3-dioxygenases (a different enzyme that catalyzes the same reaction), supported its identity as TDO. |
| GO:0006569 L-tryptophan catabolic process | IEA GO_REF:0000117 | ACCEPT | Summary: L-tryptophan catabolic process is annotated via ARBA machine learning. TDO catalyzes the oxidative cleavage of L-tryptophan as the first step in the kynurenine pathway. Crawford et al. (2020) showed that TDO knockout eliminates ommochrome biosynthesis from tryptophan in D. pealeii [PMID:32735817]. This term is correct but could be more specific. The UniProt record lists both GO:0019441 (L-tryptophan catabolic process to kynurenine) and GO:0019442 (L-tryptophan catabolic process to acetyl-CoA), and GO:0019441 more precisely captures TDO function as it catalyzes the first step to kynurenine specifically. Reason: L-tryptophan catabolic process correctly describes the biological process TDO participates in. While a more specific term like L-tryptophan catabolic process to kynurenine (GO:0019441) would be ideal, that term is now obsolete in GO. GO:0006569 is the most appropriate active term for this function. Crawford et al. (2020) showed TDO knockout eliminates the tryptophan-to-ommochrome pathway in D. pealeii [PMID:32735817]. Supporting Evidence: PMID:32735817 TDO catalyzes the first committed step in ommochrome biosynthesis, converting Tryptophan to N-Formylkyneurenine. |
| GO:0020037 heme binding | IEA GO_REF:0000120 | ACCEPT | Summary: Heme binding is annotated by combined IEA methods (InterPro, ARBA, PANTHER, UniRule). The UniProt record explicitly states: "Binds 1 heme group per subunit" (HAMAP-Rule:MF_03020), and lists heme as a cofactor (ChEBI:30413). TDO is a heme-dependent dioxygenase, and heme is essential for its catalytic mechanism. This is well-established for TDO family members across all species. Reason: Heme binding is integral to TDO catalytic function. The enzyme requires bound heme to catalyze oxidative cleavage of the tryptophan indole ring. This is confirmed by UniProt cofactor annotation and is a defining feature of the TDO family (HAMAP MF_03020, Pfam PF03301 Trp_dioxygenase). Supporting Evidence: PMID:32735817 we determined whether a TDO-selective inhibitor (680C91) impeded pigmentation in developing embryos. |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Metal ion binding is annotated via InterPro2GO mapping (IPR037217). This is correct as TDO binds iron through its heme prosthetic group. However, this term is very generic and redundant with the more informative heme binding (GO:0020037) annotation already present, which more precisely captures the nature of the metal cofactor interaction. Reason: While technically correct (TDO binds iron via heme), GO:0046872 (metal ion binding) is too generic to be informative. The heme binding annotation (GO:0020037) already captures this function at a more appropriate level of specificity. The metal ion binding term does not add useful functional information beyond what heme binding conveys. |
| GO:0006727 ommochrome biosynthetic process | IDA PMID:32735817 Highly Efficient Knockout of a Squid Pigmentation Gene | NEW | Summary: NEW annotation. Crawford et al. (2020) demonstrated by CRISPR-Cas9 knockout that TDO is required for ommochrome biosynthesis in D. pealeii. TDO knockout eliminated pigmentation in both chromatophores and eyes, which contain ommochrome pigments. The TDO-selective inhibitor 680C91 also blocked pigmentation. In situ hybridization confirmed TDO expression in chromatophore precursors and eyes. This is the most biologically meaningful annotation for this gene in cephalopods, as ommochrome pigmentation is the primary downstream biological process served by TDO in this organism. Reason: Ommochrome biosynthesis is the key biological process requiring TDO in cephalopods. CRISPR knockout and pharmacological inhibition both demonstrate TDO is essential for ommochrome production. This annotation is not present in the existing GOA set but is strongly supported by direct experimental evidence in this species. Supporting Evidence: PMID:32735817 In cephalopods, the pigments in the eyes and chromatophores are ommochromes, a derivative of tryptophan PMID:32735817 Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. PMID:32735817 We added 680C91 to developing embryos at stage 20, and it clearly blocked pigmentation in both the eyes and chromatophores, with animals developing normally otherwise. PMID:29989284 Ommochromes are widely occurring coloured molecules of invertebrates, arising from tryptophan catabolism through the so-called Tryptophan → Ommochrome pathway. |
| GO:0043473 pigmentation | IMP PMID:32735817 Highly Efficient Knockout of a Squid Pigmentation Gene | NEW | Summary: NEW annotation. Crawford et al. (2020) showed that TDO knockout by CRISPR-Cas9 resulted in loss of pigmentation in D. pealeii embryos, with chromatophore and eye pigmentation eliminated. Ink sac melanin was unaffected, demonstrating TDO specifically contributes to ommochrome-based pigmentation. Ahuja et al. (2023) further showed in E. berryi that TDO knockout delays pigmentation and TDO+IDO double knockout produces complete albinism [PMID:37343558]. Reason: Pigmentation is the primary phenotype affected by TDO loss of function. The knockout phenotype directly demonstrates involvement in pigmentation. This is a core biological role for TDO in cephalopods, where ommochrome pigments color the chromatophores and retina. Supporting Evidence: PMID:32735817 pigmentation is completely absent from the chromatophores and is minimal in the eyes which are light red PMID:37343558 The knockout of tdo in D. pealeii completely inhibited pigmentation but had little to no effect in E. berryi. |
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Download this section (compressed HTML)Q: Does D. pealeii TDO have any role in tryptophan catabolism beyond ommochrome biosynthesis (e.g. kynurenine pathway metabolites for immune or neural signaling)?
Suggested experts: Rosenthal JJC, Albertin CB
Q: What are the consequences of partial TDO loss on chromatophore function and dynamic color change in adult squid?
Suggested experts: Crawford K
Q: Is there any post-translational regulation of TDO activity in cephalopods, as seen with substrate activation in mammalian TDO?
Experiment: Characterize the enzymatic kinetics of recombinant D. pealeii TDO, including heme stoichiometry and substrate specificity, to confirm in vitro activity.
Hypothesis: D. pealeii TDO has similar catalytic properties to mammalian TDO, with strict specificity for L-tryptophan over other indoleamine substrates.
Type: biochemistry
Experiment: Use single-cell RNA-seq to map TDO expression across chromatophore subtypes and developmental stages in D. pealeii embryos.
Hypothesis: TDO expression is restricted to ommochrome-producing cell types (chromatophores and retinal pigment cells) and onset correlates with pigmentation timing.
Type: transcriptomics
Experiment: Measure kynurenine pathway metabolite levels (N-formylkynurenine, kynurenine, 3-hydroxykynurenine, xanthurenic acid) in TDO knockout vs wildtype D. pealeii embryos by LC-MS/MS.
Hypothesis: TDO knockout eliminates kynurenine pathway flux entirely in D. pealeii due to the absence of IDO, unlike in E. berryi where IDO provides compensation.
Type: metabolomics
Experiment: Test whether partial TDO knockout in adult squid affects chromatophore expansion dynamics and camouflage behavior using video analysis.
Hypothesis: Reduced ommochrome content from partial TDO loss impairs chromatophore opacity and dynamic range of color change.
Type: behavioral/physiological
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