TDO (Tryptophan 2,3-dioxygenase, AGAP002721) is the sole enzyme initiating L-tryptophan degradation via the kynurenine pathway in Anopheles gambiae. It is a heme-dependent dioxygenase that catalyzes the oxidative cleavage of the indole ring of L-tryptophan to produce N-formylkynurenine, the first and rate-limiting step of tryptophan catabolism. TDO is a homolog of the Drosophila vermilion gene and is essential for ommochrome eye pigment biosynthesis in insects. The gene maps to chromosome 2R (inside inversion 2Rb), encodes a 392 amino acid protein that assembles as a homotetramer, and is ubiquitously expressed across tissues with developmental regulation. Its enzymatic product feeds into the kynurenine pathway, which in mosquitoes is critical for processing tryptophan from blood meals, producing xanthurenic acid (a trigger of Plasmodium gametogenesis in the midgut), and maintaining midgut homeostasis. The A. gambiae TDO cDNA functionally complements Drosophila vermilion eye color mutants, confirming conserved function. The gene also serves as a visible phenotypic marker for mosquito germline transformation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0004833
L-tryptophan 2,3-dioxygenase activity
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: This IBA annotation is derived from phylogenetic inference, with supporting evidence from Drosophila vermilion (FBgn0003965) and other TDO orthologs. The A. gambiae protein shares 74% identity with Drosophila TDO and functional complementation has been demonstrated experimentally.
Reason: TDO activity is the core molecular function of this protein. The phylogenetic inference is strongly supported by sequence conservation (74% identity to Drosophila), functional complementation of Drosophila vermilion mutants (PMID:9443379), and direct biochemical characterization of recombinant A. gambiae TDO showing Michaelis-Menten kinetics (PMID:18687401).
Supporting Evidence:
PMID:18687401
In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway
PMID:9443379
The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product
file:ANOGA/TDO/TDO-deep-research-bioreason-sft.md
the active-site geometry and redox environment enable GO:0004833 tryptophan 2,3-dioxygenase activity
|
|
GO:0020037
heme binding
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: This IBA annotation for heme binding is inferred from phylogenetic analysis and supported by the conserved heme-binding residue (His-313 axial ligand) and direct spectral analysis of the recombinant protein.
Reason: Heme binding is integral to TDO catalysis. Paglino et al. (2008) purified recombinant A. gambiae TDO in its heme-ferric form and performed spectral analyses confirming heme incorporation. The active-site histidine serving as the proximal axial ligand for the heme iron is conserved.
Supporting Evidence:
PMID:18687401
Spectral analyses showed that the enzyme was purified in its heme-ferric form
|
|
GO:0004833
L-tryptophan 2,3-dioxygenase activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: Redundant IEA annotation for TDO activity derived from combined automated methods (ARBA, InterPro, RHEA, EC number, UniRule). Correct but redundant with the IBA annotation above.
Reason: The annotation is correct. TDO activity is experimentally validated by biochemical characterization (PMID:18687401) and functional complementation (PMID:9443379). Redundancy with IBA is expected and acceptable.
Supporting Evidence:
PMID:18687401
TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring
|
|
GO:0006727
ommochrome biosynthetic process
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This IEA annotation places TDO in the ommochrome biosynthetic pathway, derived from UniRule mapping. TDO catalyzes the first committed step of ommochrome biosynthesis by diverting tryptophan into the kynurenine pathway.
Reason: TDO is the first enzyme in the ommochrome biosynthetic pathway in insects. The Drosophila vermilion mutant (TDO loss-of-function) lacks ommochrome eye pigments. Functional complementation of vermilion by A. gambiae TDO confirms its role in ommochrome biosynthesis (PMID:9443379). Multiple studies on insect ommochrome pathways confirm TDO as the initiating step.
Supporting Evidence:
PMID:8969464
Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation
PMID:9443379
The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color
|
|
GO:0020037
heme binding
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: Redundant IEA annotation for heme binding from combined automated methods. Correct but redundant with the IBA annotation.
Reason: Correct. Heme binding is experimentally confirmed by spectral analysis of recombinant A. gambiae TDO (PMID:18687401).
Supporting Evidence:
PMID:18687401
Spectral analyses showed that the enzyme was purified in its heme-ferric form
|
|
GO:0046872
metal ion binding
|
IEA
GO_REF:0000002 |
KEEP AS NON CORE |
Summary: This IEA annotation for metal ion binding is derived from InterPro domain mapping (IPR037217, Tryptophan/Indoleamine 2,3-dioxygenase-like superfamily). TDO binds iron as part of its heme prosthetic group.
Reason: Technically correct because TDO binds heme iron, but the term is overly general. The more specific GO:0020037 (heme binding) already captures this function accurately. Metal ion binding is not independently informative for this protein.
Supporting Evidence:
PMID:18687401
Spectral analyses showed that the enzyme was purified in its heme-ferric form
|
|
GO:0004833
L-tryptophan 2,3-dioxygenase activity
|
ISS
GO_REF:0000024 |
ACCEPT |
Summary: ISS annotation for TDO activity based on manual transfer from Q17P71 (Aedes aegypti TDO) by curator judgment. Redundant with IBA and IEA annotations but independently validated.
Reason: Correct and well-supported. The ISS reference organism (Aedes aegypti) is a closely related mosquito. Direct biochemical evidence exists for A. gambiae TDO (PMID:18687401).
Supporting Evidence:
PMID:18687401
TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring
|
|
GO:0006569
L-tryptophan catabolic process
|
ISS
GO_REF:0000024 |
ACCEPT |
Summary: ISS annotation for L-tryptophan catabolic process transferred from Aedes aegypti TDO. TDO catalyzes the first step of tryptophan catabolism.
Reason: TDO is the sole enzyme initiating tryptophan catabolism in A. gambiae (PMID:18687401). The annotation is appropriate as TDO commits tryptophan irreversibly to catabolism. The term GO:0006569 is suitably general for what TDO does (initiates catabolism) without over-specifying the downstream fate of the carbon.
Supporting Evidence:
PMID:18687401
In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway
|
|
GO:0020037
heme binding
|
ISS
GO_REF:0000024 |
ACCEPT |
Summary: ISS annotation for heme binding transferred from Aedes aegypti TDO. Redundant with IBA and IEA annotations but correct.
Reason: Correct. Heme binding is experimentally confirmed by spectral analysis of recombinant A. gambiae TDO (PMID:18687401).
Supporting Evidence:
PMID:18687401
Spectral analyses showed that the enzyme was purified in its heme-ferric form
|
Q: Does A. gambiae TDO expression change in response to blood feeding, and if so, what is the tissue-specific and temporal pattern of induction?
Q: What are the kinetic parameters (Km, kcat) of A. gambiae TDO for L-tryptophan under physiologically relevant reductant conditions?
Q: Does TDO disruption in A. gambiae affect Plasmodium transmission by altering xanthurenic acid levels in the midgut?
Experiment: CRISPR knockout of TDO in A. gambiae to assess effects on eye pigmentation, blood meal processing, survival, fecundity, and Plasmodium transmission.
Hypothesis: TDO knockout will produce white/vermilion-like eyes, impair tryptophan homeostasis after blood feeding, and alter Plasmodium development in the midgut through changes in xanthurenic acid production.
Experiment: Tissue-specific and blood meal-responsive transcriptomic profiling of TDO and downstream kynurenine pathway genes in A. gambiae.
Hypothesis: TDO expression is induced in the midgut following blood feeding to process the tryptophan load from hemoglobin digestion.
I begin with the InterPro architecture. Three overlapping family signatures—IPR004981 (Tryptophan 2,3-dioxygenase family) spanning residues 8–378, 11–358, and 20–358—enclose a core catalytic region that is further encompassed by IPR037217 (Tryptophan/Indoleamine 2,3-dioxygenase-like homologous superfamily) from residues 27–359. This layered arrangement is characteristic of heme-dependent tryptophan/indoleamine 2,3-dioxygenases: a conserved catalytic fold that binds a b-type heme via a proximal histidine and positions molecular oxygen for insertion into the indole ring of L-tryptophan. The breadth of the family signatures across the central polypeptide indicates a single-domain enzyme whose N- and C-termini contribute to oligomerization interfaces rather than distinct modules. Such enzymes typically assemble into higher-order complexes (often tetramers), and the conserved fold stabilizes the heme pocket and the dioxygen activation chemistry.
This architecture causes two molecular functions. First, the heme pocket formed by the conserved fold enforces GO:0020037 heme binding. Second, the active-site geometry and redox environment enable GO:0004833 tryptophan 2,3-dioxygenase activity, in which O2 is inserted across the C2–C3 bond of the indole ring to generate N-formylkynurenine. The catalytic outcome is mechanistically inseparable from a heme–O2 adduct that is subsequently resolved by electron input from a reductant, consistent with the requirement for a physiological reductant such as NADPH via an associated reductase.
The immediate biochemical consequence of this activity is the first committed step of the kynurenine pathway, producing N-formylkynurenine that is then hydrolyzed to kynurenine. This places the enzyme squarely in GO:0019441 tryptophan catabolic process to kynurenine. Because the reaction irreversibly diverts L-tryptophan from biosynthesis and signaling roles into catabolism, the enzyme functions as a metabolic gatekeeper controlling flux into downstream kynurenine- and NAD+–related branches.
The oligomeric nature implied by the family architecture supports assembly into a GO:1902494 catalytic complex, where subunits cooperate to stabilize the heme environment and coordinate access to substrate and reductant. The absence of transmembrane segments and the soluble enzyme fold argue for a cytosolic location, where substrate availability and redox partners are abundant; thus a cytosolic catalytic complex is the most plausible operational site.
From this mechanistic base, I hypothesize interaction partners and pathway wiring. Kynurenine formamidase likely associates functionally to receive N-formylkynurenine or kynurenine, creating a substrate channel that minimizes diffusion and protects labile intermediates. Kynurenine 3-monooxygenase would engage downstream to convert kynurenine toward 3-hydroxykynurenine, linking to NAD+ biosynthesis and oxidative stress chemistry. Tyrosine aminotransferase may intersect at points of aromatic amino acid balance, drawing on kynurenine-derived pools or competing for shared cofactors and redox currency. Hormone-sensitive lipase could modulate local lipid-derived NADPH production or membrane-proximal redox microenvironments that influence the reductase-dependent step. The phosphotransferase LOC123688 and several Anopheles gene products (AGAP006020-PA., AGAP009091-PA., AGAP011158-PA., AGAP004500-PA., AGAP003887-PB.) plausibly act as regulatory or scaffolding factors that tune activity via phosphorylation, complex assembly, or localization, thereby coupling tryptophan catabolism to developmental or stress-responsive programs in the mosquito.
## Functional Summary
A heme-dependent dioxygenase in the African malaria mosquito that assembles into a soluble catalytic complex and catalyzes the first, rate-setting step of tryptophan breakdown by inserting oxygen across the indole ring of L-tryptophan to form N-formylkynurenine. The reaction requires a physiological reductant, consistent with electron transfer to the heme–oxygen intermediate during catalysis. By committing tryptophan to the kynurenine pathway, it governs flux into downstream catabolic branches, with proximal enzymes likely associating to channel intermediates and coordinate redox supply in the cytosol.
## UniProt Summary
Heme-dependent dioxygenase that catalyzes the first and rate-limiting step of the catabolism of the essential amino acid tryptophan along the kynurenine pathway. Catalyzes the oxidative cleavage of the L-tryptophan indole ring and converts L-tryptophan to N-formylkynurenine. Requires a reductant to be physically and functionally incorporated in the catalytic cycle (By similarity).
## InterPro Domains
- IPR004981: Tryptophan 2,3-dioxygenase (family) [8-378]
- IPR004981: Tryptophan 2,3-dioxygenase (family) [11-358]
- IPR004981: Tryptophan 2,3-dioxygenase (family) [20-358]
- IPR037217: Tryptophan/Indoleamine 2,3-dioxygenase-like (homologous_superfamily) [27-359]
## GO Term Predictions
### Molecular Function
### Biological Process
### Cellular Component
The foundational paper. Cloned and characterized the A. gambiae tryptophan oxygenase gene (homolog of Drosophila vermilion).
- Maps to chromosome 2R, subdivision 12E (unlike Drosophila where it is X-linked)
- Six introns: four positioned identically to Drosophila, one similarly positioned, one novel
- 1955 nt cDNA encoding 392 amino acid protein (~45 kDa)
- 74% identity to Drosophila, 53% identity to nematode/mammalian TDOs
- Developmentally regulated transcript ~2 kb
- "Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation" PMID:8969464
The key biochemical paper on recombinant A. gambiae TDO.
- "In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway" PMID:18687401
- TDO mRNA is ubiquitously expressed across tissues
- Purified recombinant enzyme in heme-ferric form
- Determined Michaelis-Menten kinetics with reducing agents
- Several kynurenines and tryptophan-derived molecules modulate TDO activity in vitro
- "Our study could contribute to understanding TDO regulation in vivo and to the identification of inhibitors to be used to alter Tryptophan homeostasis in the malaria vector" PMID:18687401
TDO catalyzes step 1: L-tryptophan + O2 --> N-formylkynurenine
N-formylkynurenine is then hydrolyzed to kynurenine by kynurenine formamidase.
Kynurenine --> 3-hydroxykynurenine (via kynurenine 3-monooxygenase)
3-HK --> xanthurenic acid (via 3-HKT) OR --> ommochrome pigments
The GOA annotations are all computational:
- IBA (phylogenetic inference): TDO activity, heme binding -- supported by Drosophila vermilion orthology
- IEA (electronic annotation): TDO activity, heme binding, ommochrome biosynthesis, metal ion binding
- ISS (sequence similarity): TDO activity, heme binding, L-tryptophan catabolism (based on Q17P71)
The UniProt record also lists GO:0019441 (L-tryptophan catabolic process to kynurenine, ISS) and GO:0019442 (L-tryptophan catabolic process to acetyl-CoA, IBA) but these were not present in the GOA download.
Note: GO:0019442 (L-tryptophan catabolic process to acetyl-CoA) is an over-annotation for this enzyme. TDO only catalyzes the first step to N-formylkynurenine. The full pathway to acetyl-CoA requires many additional enzymes. GO:0019441 (to kynurenine) is more appropriate but still covers two enzymatic steps (TDO + formamidase).
The BioReason deep-research file correctly identifies:
- Heme-dependent dioxygenase activity
- Kynurenine pathway first step
- Heme binding
- Homotetramer assembly
Issues identified:
1. No literature citations in the trace -- all from InterPro/UniProt
2. Speculative interaction partners: "Hormone-sensitive lipase could modulate local lipid-derived NADPH production" -- no evidence
3. Claims about "GO:1902494 catalytic complex" -- no experimental evidence for TDO complex annotation
4. Mentions of AGAP006020-PA, AGAP009091-PA etc. as "regulatory or scaffolding factors" -- speculative STRING neighbors
5. The thinking trace is reasonable but generic; misses the organism-specific biology (eye pigmentation, blood meal processing, malaria transmission link)
6. No GO predictions listed in the MF/BP/CC sections despite discussing functions in the thinking trace
Source: TDO-deep-research-bioreason-sft.md
The BioReason SFT functional summary states:
A heme-dependent dioxygenase in the African malaria mosquito that assembles into a soluble catalytic complex and catalyzes the first, rate-setting step of tryptophan breakdown by inserting oxygen across the indole ring of L-tryptophan to form N-formylkynurenine. The reaction requires a physiological reductant, consistent with electron transfer to the heme-oxygen intermediate during catalysis. By committing tryptophan to the kynurenine pathway, it governs flux into downstream catabolic branches, with proximal enzymes likely associating to channel intermediates and coordinate redox supply in the cytosol.
The core enzymatic description is accurate: heme-dependent dioxygenase, first step of kynurenine pathway, L-tryptophan to N-formylkynurenine, requires reductant. This aligns with the biochemical characterization by Paglino et al. (2008, PMID:18687401).
Errors and issues:
"Soluble catalytic complex" is speculative: The thinking trace predicts "GO:1902494 catalytic complex" and claims subunits "cooperate to stabilize the heme environment." While TDO is indeed a homotetramer (dimer of dimers), there is no evidence for GO:1902494 annotation. The homotetrameric assembly is well-established for TDO family members, but the "catalytic complex" framing implies a multi-protein complex rather than homo-oligomerization.
Speculative interaction partners: The thinking trace lists several hypothetical interaction partners: "Kynurenine formamidase likely associates functionally," "Hormone-sensitive lipase could modulate local lipid-derived NADPH production," and several Anopheles gene products (AGAP006020-PA, AGAP009091-PA, AGAP011158-PA, AGAP004500-PA, AGAP003887-PB) described as "regulatory or scaffolding factors." None of these interactions have experimental support. The interaction partners appear to be STRING database co-expression neighbors presented as functionally validated associations.
"Proximal enzymes likely associating to channel intermediates" is unsupported: The claim of substrate channeling between TDO and kynurenine formamidase has no experimental evidence in any organism, and is speculative.
What is missing (completeness gaps):
Ommochrome biosynthesis and eye pigmentation completely absent: The most biologically distinctive function of insect TDO -- its role as the first enzyme in ommochrome pigment biosynthesis and its control of eye color (the vermilion phenotype) -- is not mentioned at all. This is the function that gives the gene its historical name and practical importance as a transformation marker. The GOA annotation GO:0006727 (ommochrome biosynthetic process) is present but BioReason does not address it.
Functional complementation of Drosophila vermilion absent: Besansky et al. (1997, PMID:9443379) demonstrated that A. gambiae TDO functionally rescues Drosophila vermilion eye color. This is direct experimental evidence for proper enzymatic function but is not referenced.
Blood meal processing absent: A critical aspect of TDO biology in hematophagous mosquitoes is processing the massive tryptophan load from blood meal hemoglobin digestion. Disruption of the kynurenine pathway causes midgut homeostasis dysfunction and reproductive costs (PMID:34999199). This organism-specific biology is entirely absent.
Malaria transmission link absent: The downstream product xanthurenic acid is a gametocyte-activating factor for Plasmodium parasites (PMID:11463462, PMID:16585514). TDO's role in the pathway that produces this malaria-relevant metabolite is a major aspect of its biological significance in A. gambiae but is not mentioned.
Ubiquitous tissue expression absent: Paglino et al. (2008, PMID:18687401) showed TDO mRNA is ubiquitously expressed across A. gambiae tissues, underscoring its metabolic importance beyond eye pigmentation. This is not mentioned.
Transformation marker utility absent: The gene's importance as a visible phenotypic marker for mosquito transgenesis (PMID:8969464) is a practical application but is absent.
No GO predictions listed: The MF, BP, and CC prediction sections are all empty despite the thinking trace discussing multiple GO terms. This appears to be a formatting or generation issue.
The thinking trace is methodical in deriving molecular function from InterPro domain architecture. The deduction of heme binding and TDO activity from the domain signatures is sound. The description of the catalytic mechanism (heme-O2 adduct, indole ring cleavage) is biochemically accurate.
However, the trace exhibits a characteristic BioReason failure mode: it builds an elaborate mechanistic narrative from domain architecture alone without consulting organism-specific literature. The result is a generic TDO description that could apply to any TDO from any organism. The unique biology of A. gambiae TDO -- ommochrome pigmentation, blood meal metabolism, malaria transmission, and transgenesis utility -- is entirely absent because these functions cannot be inferred from InterPro domains alone.
The phosphotransferase "LOC123688" mentioned as an interaction partner is not a recognized A. gambiae gene identifier, suggesting confusion with a different organism's gene nomenclature.
The InterPro2GO annotations provide:
- GO:0046872 (metal ion binding) from IPR037217
BioReason correctly identifies TDO activity and heme binding from the domain architecture, going beyond what InterPro2GO captures. This is a case where BioReason adds genuine value by correctly interpreting the TDO-specific family signature (IPR004981). However, the organism-specific biological context is absent from both sources.
The IBA annotations (GO:0004833, GO:0020037) and the UniRule-derived GO:0006727 (ommochrome biosynthesis) capture the protein's biology more accurately than BioReason's output, which misses the ommochrome pathway entirely despite it being the most distinctive biological role of insect TDO.
The BioReason trace contains no literature citations (no PMIDs). All functional inferences derive from InterPro domain annotations and the UniProt summary text. The "UniProt Summary" section in the BioReason output accurately reflects the actual UniProt record, so no fabrication is detected in this case (unlike some other BioReason outputs). The trace is honest in that it does not cite papers it has not read, but this means all organism-specific knowledge is absent.
id: O77457
gene_symbol: TDO
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:7165
label: Anopheles gambiae
description: >-
TDO (Tryptophan 2,3-dioxygenase, AGAP002721) is the sole enzyme initiating
L-tryptophan degradation via the kynurenine pathway in Anopheles gambiae. It is
a heme-dependent dioxygenase that catalyzes the oxidative cleavage of the indole
ring of L-tryptophan to produce N-formylkynurenine, the first and rate-limiting
step of tryptophan catabolism. TDO is a homolog of the Drosophila vermilion gene
and is essential for ommochrome eye pigment biosynthesis in insects. The gene maps
to chromosome 2R (inside inversion 2Rb), encodes a 392 amino acid protein that
assembles as a homotetramer, and is ubiquitously expressed across tissues with
developmental regulation. Its enzymatic product feeds into the kynurenine pathway,
which in mosquitoes is critical for processing tryptophan from blood meals,
producing xanthurenic acid (a trigger of Plasmodium gametogenesis in the midgut),
and maintaining midgut homeostasis. The A. gambiae TDO cDNA functionally
complements Drosophila vermilion eye color mutants, confirming conserved function.
The gene also serves as a visible phenotypic marker for mosquito germline
transformation.
existing_annotations:
- term:
id: GO:0004833
label: L-tryptophan 2,3-dioxygenase activity
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
This IBA annotation is derived from phylogenetic inference, with supporting
evidence from Drosophila vermilion (FBgn0003965) and other TDO orthologs.
The A. gambiae protein shares 74% identity with Drosophila TDO and functional
complementation has been demonstrated experimentally.
action: ACCEPT
reason: >-
TDO activity is the core molecular function of this protein. The phylogenetic
inference is strongly supported by sequence conservation (74% identity to
Drosophila), functional complementation of Drosophila vermilion mutants
(PMID:9443379), and direct biochemical characterization of recombinant
A. gambiae TDO showing Michaelis-Menten kinetics (PMID:18687401).
additional_reference_ids:
- PMID:9443379
- PMID:18687401
supported_by:
- reference_id: PMID:18687401
supporting_text: "In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway"
- reference_id: PMID:9443379
supporting_text: "The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product"
- reference_id: file:ANOGA/TDO/TDO-deep-research-bioreason-sft.md
supporting_text: "the active-site geometry and redox environment enable GO:0004833 tryptophan 2,3-dioxygenase activity"
- term:
id: GO:0020037
label: heme binding
evidence_type: IBA
original_reference_id: GO_REF:0000033
review:
summary: >-
This IBA annotation for heme binding is inferred from phylogenetic analysis
and supported by the conserved heme-binding residue (His-313 axial ligand)
and direct spectral analysis of the recombinant protein.
action: ACCEPT
reason: >-
Heme binding is integral to TDO catalysis. Paglino et al. (2008) purified
recombinant A. gambiae TDO in its heme-ferric form and performed spectral
analyses confirming heme incorporation. The active-site histidine serving
as the proximal axial ligand for the heme iron is conserved.
additional_reference_ids:
- PMID:18687401
supported_by:
- reference_id: PMID:18687401
supporting_text: "Spectral analyses showed that the enzyme was purified in its heme-ferric form"
- term:
id: GO:0004833
label: L-tryptophan 2,3-dioxygenase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
Redundant IEA annotation for TDO activity derived from combined automated
methods (ARBA, InterPro, RHEA, EC number, UniRule). Correct but redundant
with the IBA annotation above.
action: ACCEPT
reason: >-
The annotation is correct. TDO activity is experimentally validated by
biochemical characterization (PMID:18687401) and functional complementation
(PMID:9443379). Redundancy with IBA is expected and acceptable.
supported_by:
- reference_id: PMID:18687401
supporting_text: "TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring"
- term:
id: GO:0006727
label: ommochrome biosynthetic process
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
This IEA annotation places TDO in the ommochrome biosynthetic pathway, derived
from UniRule mapping. TDO catalyzes the first committed step of ommochrome
biosynthesis by diverting tryptophan into the kynurenine pathway.
action: ACCEPT
reason: >-
TDO is the first enzyme in the ommochrome biosynthetic pathway in insects.
The Drosophila vermilion mutant (TDO loss-of-function) lacks ommochrome eye
pigments. Functional complementation of vermilion by A. gambiae TDO confirms
its role in ommochrome biosynthesis (PMID:9443379). Multiple studies on
insect ommochrome pathways confirm TDO as the initiating step.
additional_reference_ids:
- PMID:9443379
- PMID:8969464
supported_by:
- reference_id: PMID:8969464
supporting_text: "Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation"
- reference_id: PMID:9443379
supporting_text: "The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color"
- term:
id: GO:0020037
label: heme binding
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
Redundant IEA annotation for heme binding from combined automated methods.
Correct but redundant with the IBA annotation.
action: ACCEPT
reason: >-
Correct. Heme binding is experimentally confirmed by spectral analysis of
recombinant A. gambiae TDO (PMID:18687401).
supported_by:
- reference_id: PMID:18687401
supporting_text: "Spectral analyses showed that the enzyme was purified in its heme-ferric form"
- term:
id: GO:0046872
label: metal ion binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This IEA annotation for metal ion binding is derived from InterPro domain
mapping (IPR037217, Tryptophan/Indoleamine 2,3-dioxygenase-like superfamily).
TDO binds iron as part of its heme prosthetic group.
action: KEEP_AS_NON_CORE
reason: >-
Technically correct because TDO binds heme iron, but the term is overly
general. The more specific GO:0020037 (heme binding) already captures this
function accurately. Metal ion binding is not independently informative for
this protein.
supported_by:
- reference_id: PMID:18687401
supporting_text: "Spectral analyses showed that the enzyme was purified in its heme-ferric form"
- term:
id: GO:0004833
label: L-tryptophan 2,3-dioxygenase activity
evidence_type: ISS
original_reference_id: GO_REF:0000024
review:
summary: >-
ISS annotation for TDO activity based on manual transfer from Q17P71
(Aedes aegypti TDO) by curator judgment. Redundant with IBA and IEA
annotations but independently validated.
action: ACCEPT
reason: >-
Correct and well-supported. The ISS reference organism (Aedes aegypti) is a
closely related mosquito. Direct biochemical evidence exists for A. gambiae
TDO (PMID:18687401).
supported_by:
- reference_id: PMID:18687401
supporting_text: "TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring"
- term:
id: GO:0006569
label: L-tryptophan catabolic process
evidence_type: ISS
original_reference_id: GO_REF:0000024
review:
summary: >-
ISS annotation for L-tryptophan catabolic process transferred from Aedes
aegypti TDO. TDO catalyzes the first step of tryptophan catabolism.
action: ACCEPT
reason: >-
TDO is the sole enzyme initiating tryptophan catabolism in A. gambiae
(PMID:18687401). The annotation is appropriate as TDO commits tryptophan
irreversibly to catabolism. The term GO:0006569 is suitably general for
what TDO does (initiates catabolism) without over-specifying the downstream
fate of the carbon.
additional_reference_ids:
- PMID:18687401
supported_by:
- reference_id: PMID:18687401
supporting_text: "In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway"
- term:
id: GO:0020037
label: heme binding
evidence_type: ISS
original_reference_id: GO_REF:0000024
review:
summary: >-
ISS annotation for heme binding transferred from Aedes aegypti TDO.
Redundant with IBA and IEA annotations but correct.
action: ACCEPT
reason: >-
Correct. Heme binding is experimentally confirmed by spectral analysis
of recombinant A. gambiae TDO (PMID:18687401).
supported_by:
- reference_id: PMID:18687401
supporting_text: "Spectral analyses showed that the enzyme was purified in its heme-ferric form"
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000024
title: Manual transfer of experimentally-verified manual GO annotation data to orthologs by curator judgment of sequence similarity
findings: []
- id: GO_REF:0000033
title: Annotation inferences using phylogenetic trees
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: PMID:8969464
title: The tryptophan oxygenase gene of Anopheles gambiae.
findings:
- statement: The A. gambiae TDO gene was cloned and maps to chromosome 2R
supporting_text: "The Anopheles gambiae gene encoding tryptophan oxygenase, a homolog of the Drosophila melanogaster vermilion gene, has been molecularly cloned and characterized"
- statement: TDO encodes a 392 amino acid protein with 74% identity to Drosophila
supporting_text: "Amino acid comparisons between the deduced protein and previously known tryptophan oxygenases revealed 74% identity between Anopheles and Drosophila"
- statement: TDO transcript is developmentally regulated
supporting_text: "Northern analysis detected a developmentally regulated transcript about 2 kb in length"
- statement: TDO controls eye color and can serve as a transformation marker
supporting_text: "Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation"
- id: PMID:9443379
title: The Anopheles gambiae tryptophan oxygenase gene expressed from a baculovirus promoter complements Drosophila melanogaster vermilion.
findings:
- statement: A. gambiae TDO functionally complements Drosophila vermilion eye color
supporting_text: "The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color"
- statement: Demonstrates proper function of the A. gambiae TDO product
supporting_text: "The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product and the effectiveness of the ie-1 promoter in directing expression of foreign genes in live insects"
- id: PMID:11240635
title: "Patterns of DNA sequence variation in chromosomally recognized taxa of Anopheles gambiae: evidence from rDNA and single-copy loci."
findings:
- statement: TDO locus inside inversion 2Rb shows introgressive hybridization between species
supporting_text: "At the tryptophan oxygenase locus inside inversion 2Rb, variation was structured only by inversion orientation and not by taxonomic designation even between An. gambiae and An. arabiensis"
- id: PMID:12364791
title: The genome sequence of the malaria mosquito Anopheles gambiae.
findings:
- statement: Provides genomic context for AGAP002721 on chromosome 2R
supporting_text: "The genome sequence of the malaria mosquito Anopheles gambiae"
- id: PMID:18687401
title: Purification and biochemical characterization of a recombinant Anopheles gambiae tryptophan 2,3-dioxygenase expressed in Escherichia coli.
findings:
- statement: TDO is the only enzyme initiating tryptophan degradation via the kynurenine pathway in A. gambiae
supporting_text: "In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway"
- statement: TDO mRNA is ubiquitously expressed across tissues
supporting_text: "a RT-PCR-based analysis of the tissue distribution of TDO mRNA in A. gambiae that revealed a ubiquitous expression of the gene"
- statement: Recombinant TDO purified in heme-ferric form with Michaelis-Menten kinetics determined
supporting_text: "Spectral analyses showed that the enzyme was purified in its heme-ferric form that was subsequently used to determining the Michaelis-Menten constants"
- statement: Several kynurenines and tryptophan metabolites modulate TDO activity
supporting_text: "The screening of a number of compounds as potential TDO modulators showed that several kynurenines and other Tryptophan-derived molecules interfere with the enzyme activity in vitro"
- id: PMID:34999199
title: Beyond the eye - Kynurenine pathway impairment causes midgut homeostasis dysfunction and survival and reproductive costs in blood-feeding mosquitoes.
findings:
- statement: Kynurenine pathway disruption impairs midgut homeostasis and reproduction in mosquitoes
supporting_text: "Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species"
- statement: Xanthurenic acid (KP end-product) rescues lethality from pathway disruption
supporting_text: "Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti"
- id: PMID:35437328
title: Anopheline mosquitoes are protected against parasite infection by tryptophan catabolism in gut microbiota.
findings:
- statement: Gut microbiota catabolize 3-HK to protect against Plasmodium infection
supporting_text: "3-HK impaired the structure of the peritrophic matrix and promoted Plasmodium berghei infection"
- id: PMID:16585514
title: Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase.
findings:
- statement: Xanthurenic acid plays a key role in Plasmodium gametogenesis in A. gambiae
supporting_text: "In Anopheles gambiae, the vector for the most deadly malaria parasite Plasmodium falciparum, xanthurenic acid (XA) plays a key role in parasite gametogenesis and fertility"
- id: PMID:11463462
title: Both mosquito-derived xanthurenic acid and a host blood-derived factor regulate gametogenesis of Plasmodium in the midgut of the mosquito.
findings:
- statement: Xanthurenic acid is a gametocyte-activating factor for Plasmodium
supporting_text: "Removal of both host and vector sources of GAF totally inhibited both exflagellation and ookinete production"
- id: file:ANOGA/TDO/TDO-deep-research-bioreason-sft.md
title: BioReason-Pro SFT prediction for A. gambiae TDO
findings:
- statement: Domain architecture consistent with heme-dependent TDO
supporting_text: "a core catalytic region that is further encompassed by IPR037217 from residues 27-359"
core_functions:
- molecular_function:
id: GO:0004833
label: L-tryptophan 2,3-dioxygenase activity
description: >-
TDO is a heme-dependent dioxygenase that catalyzes the oxidative cleavage of the
L-tryptophan indole ring to produce N-formylkynurenine. It is the sole enzyme
initiating tryptophan catabolism via the kynurenine pathway in A. gambiae.
Recombinant enzyme has been biochemically characterized with Michaelis-Menten
kinetics determined. The enzyme requires a physiological reductant and the
heme-ferric form is catalytically active after reduction.
directly_involved_in:
- id: GO:0006569
label: L-tryptophan catabolic process
- id: GO:0006727
label: ommochrome biosynthetic process
supported_by:
- reference_id: PMID:18687401
supporting_text: "TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring"
- reference_id: PMID:9443379
supporting_text: "The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product"
proposed_new_terms: []
suggested_questions:
- question: >-
Does A. gambiae TDO expression change in response to blood feeding, and if so,
what is the tissue-specific and temporal pattern of induction?
- question: >-
What are the kinetic parameters (Km, kcat) of A. gambiae TDO for L-tryptophan
under physiologically relevant reductant conditions?
- question: >-
Does TDO disruption in A. gambiae affect Plasmodium transmission by altering
xanthurenic acid levels in the midgut?
suggested_experiments:
- description: >-
CRISPR knockout of TDO in A. gambiae to assess effects on eye pigmentation,
blood meal processing, survival, fecundity, and Plasmodium transmission.
hypothesis: >-
TDO knockout will produce white/vermilion-like eyes, impair tryptophan
homeostasis after blood feeding, and alter Plasmodium development in the
midgut through changes in xanthurenic acid production.
- description: >-
Tissue-specific and blood meal-responsive transcriptomic profiling of TDO
and downstream kynurenine pathway genes in A. gambiae.
hypothesis: >-
TDO expression is induced in the midgut following blood feeding to process
the tryptophan load from hemoglobin digestion.