PP_4983 is an exported FAD-dependent amine oxidase family protein in Pseudomonas putida KT2440. The locus is often discussed under the alias iaaM because UniProt/GO automation assigns tryptophan 2-monooxygenase activity and auxin biosynthesis to it, but the specific iaaM interpretation is weak. The same locus is annotated by KEGG as a putative amine oxidase / monoamine oxidase (K00274, EC 1.4.3.4), and KT2440 already contains distinct amino oxidase family members including davB/PP_0383. The best current synthesis is monoamine-oxidase-like oxidoreductase activity with unresolved physiological substrate, rather than a confirmed indole-3-acetamide auxin-biosynthetic enzyme.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0016491
oxidoreductase activity
|
IEA
GO_REF:0000120 |
MARK AS OVER ANNOTATED |
Summary: PP_4983 is clearly an oxidoreductase-family protein, but this parent term is too broad to be useful. Both UniProt and KEGG place the protein within flavin-dependent amine oxidation chemistry, so a more specific amine-oxidase-like molecular function is preferable.
Reason: The broad oxidoreductase root term adds little information once the locus is interpreted as a monoamine-oxidase-like flavoprotein. It is directionally correct but not an informative core annotation.
Supporting Evidence:
file:PSEPK/iaaM/iaaM-notes.md
The safest current synthesis is amine oxidase / monoamine oxidase-like flavoprotein rather than confirmed tryptophan 2-monooxygenase in auxin biosynthesis.
|
|
GO:0050361
tryptophan 2-monooxygenase activity
|
IEA
GO_REF:0000003 |
MODIFY |
Summary: This specific iaaM-style annotation is not well supported for PP_4983. The UniProt assignment is homology-based only, whereas KEGG classifies the same locus as a putative amine oxidase / monoamine oxidase and not as a dedicated indole-3-acetamide auxin enzyme. In the same genome, davB/PP_0383 is already assigned a distinct amino-oxidase chemistry, reinforcing that automatic family transfer is likely overspecific here.
Reason: The protein is probably an amine oxidase, but the specific substrate assignment to tryptophan-to-indole-3-acetamide conversion is weaker than the broader monoamine oxidase interpretation from KEGG. Until biochemical data establish IAM formation in KT2440, the safer molecular function is monoamine oxidase activity.
Proposed replacements:
monoamine oxidase activity
Supporting Evidence:
file:PSEPK/iaaM/iaaM-uniprot.txt
Belongs to the tryptophan 2-monooxygenase family.
file:PSEPK/iaaM/iaaM-uniprot.txt
PE 3: Inferred from homology;
file:PSEPK/iaaM/iaaM-notes.md
KEGG contradicts the specific iaaM call for the same locus and instead labels PP_4983 as putative amine oxidase with orthology K00274 monoamine oxidase [EC:1.4.3.4].
|
Q: Is PP_4983 able to convert L-tryptophan to indole-3-acetamide in vitro or in vivo, or is its physiological substrate a different monoamine?
Q: Does KT2440 encode a bona fide iaaH-like amidase that pairs with PP_4983 in an indole-3-acetamide pathway?
Experiment: Purify PP_4983 and test FAD-dependent oxidation of L-tryptophan, methylamine, phenethylamine, tyramine, and related amines by LC-MS or targeted product assays. Compare turnover against authentic indole-3-acetamide production.
Hypothesis: PP_4983 is a monoamine oxidase-like flavoprotein rather than a dedicated iaaM tryptophan 2-monooxygenase.
Type: biochemical enzyme assay
Experiment: Construct a PP_4983 deletion strain and quantify indole-3-acetamide and indole-3-acetic acid in wild type and mutant cultures grown with supplemental tryptophan.
Hypothesis: If PP_4983 participates in IAM/IAA biosynthesis, deleting the locus should reduce indole-3-acetamide or indole-3-acetic acid accumulation from tryptophan.
Type: genetics plus metabolomics
Experiment: Compare PP_4983, PP_4155, and davB mutant growth and transcriptional responses across candidate amine and amino-acid substrates to separate auxin-pathway and general amine-catabolic roles.
Hypothesis: PP_4983 and the other KT2440 amine oxidase candidates have distinct substrate scopes.
Type: comparative genetics and physiology
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
UniProt accession Q88D45 (ordered locus PP_4983) in Pseudomonas putida KT2440 is annotated as tryptophan 2-monooxygenase (EC 1.13.12.3) and placed in the tryptophan 2-monooxygenase (IaaM/TMO) family, a flavin-dependent enzyme classically associated with the indole-3-acetamide (IAM) pathway for bacterial auxin (IAA) biosynthesis. However, literature retrieved here did not provide KT2440- and PP_4983-specific experimental evidence confirming that this particular KT2440 protein catalyzes L-tryptophan → IAM in vivo or in vitro.
A key disambiguation point is that authoritative synthesis work on bacterial IAA pathways reports that some P. putida KT2440 “iaaM-like” homologs fall into a divergent clade and that a KT2440 representative (named DavB in that review context) has been experimentally linked to lysine degradation rather than confirmed IAA biosynthesis, underscoring that the symbol iaaM can be misleading in P. putida unless validated at the locus/protein level. (duca2014indole3aceticacidin pages 8-9)
Accordingly, the functional discussion below is split into (i) high-confidence information about the IaaM/TMO enzyme family (EC 1.13.12.3) from primary biochemical studies and recent engineering work, and (ii) careful inference for Q88D45/PP_4983 based on that family context, explicitly noting what is and is not demonstrated for KT2440.
In the canonical IAM pathway of bacterial auxin biosynthesis, L-tryptophan is converted to indole-3-acetamide (IAM) by tryptophan 2-monooxygenase (IaaM/TMO), and IAM is hydrolyzed to indole-3-acetic acid (IAA) by an IAM hydrolase (IaaH). (duca2014indole3aceticacidin pages 8-9, duca2020indole3aceticacidbiosynthesis pages 8-9)
The genetic assignment of these enzymes in plant-pathogenic Pseudomonas has been established at the operon level: iaaM encodes tryptophan 2-monooxygenase (EC 1.13.12.3) and iaaH encodes indoleacetamide (IAM) hydrolase. (yamada1985nucleotidesequencesof pages 1-2)
In Pseudomonas syringae subsp. savastanoi (historical “P. savastanoi”), iaaM and iaaH are adjacent and cotranscribed: Northern blots detect a shared ~4–5 kb transcript; insertions in iaaM have strong polar effects on iaaH expression and produce truncated transcripts, indicating a polycistronic operon with iaaH promoter-distal. (palm1989cotranscriptionofgenes pages 5-6, palm1989cotranscriptionofgenes pages 5-5)
Sequence-level work similarly supports operon coupling: insertions in iaaM produce polar effects on iaaH, consistent with shared transcriptional control via the iaaM promoter. (yamada1985nucleotidesequencesof pages 3-4)
A highly cited synthesis of IAA pathways in plant–microbe systems reports that iaaM homologs separate into groups, with only one group corresponding to confirmed IAM-pathway IAA synthesis, whereas other homologs are diverse and may have different substrates/roles; the same source specifically cautions that a P. putida KT2440 representative (referred to as DavB) was associated with lysine degradation, not confirmed IAA synthesis. (duca2014indole3aceticacidin pages 8-9)
Primary biochemical characterization in P. syringae pv. savastanoi established that tryptophan 2-monooxygenase catalyzes oxidative decarboxylation of L-tryptophan to indole-3-acetamide (IAM) with concomitant O2 uptake; this is the first committed step in the IAM→IAA route. (hutcheson1985regulationof3indoleacetic pages 1-2, hutcheson1985regulationof3indoleacetic pages 4-5)
A recent enzyme-engineering study (2024) likewise defines TMO as an FAD-bound flavoenzyme catalyzing L-tryptophan + O2 → IAM + CO2, explicitly framing it as the first step of IAA biosynthesis via IAM. (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 2-4)
Tryptophan 2-monooxygenase is a flavoprotein containing one FAD per ~62 kDa subunit in the classic Pseudomonas system. (hutcheson1985regulationof3indoleacetic pages 1-2, hutcheson1985regulationof3indoleacetic pages 4-5)
The 2024 engineering paper uses spectroscopic monitoring consistent with enzyme-bound FAD (e.g., absorbance at 466 nm) and explicitly models the enzyme with bound FAD in molecular dynamics analyses. (kongjaroon2024enhancementoftryptophan pages 9-10, kongjaroon2024enhancementoftryptophan pages 11-12)
The purified P. savastanoi tryptophan 2-monooxygenase accepts multiple substituted tryptophans (e.g., 4–7 methyl-tryptophan, 7-aza-tryptophan, 5-fluoro-tryptophan, 5-hydroxy-tryptophan) but does not accept D-tryptophan or α-methyltryptophan, indicating stereospecificity and constraints on α-carbon substitution. (hutcheson1985regulationof3indoleacetic pages 4-5)
For the P. savastanoi enzyme, apparent kinetic parameters include Km(L-Trp) ≈ 50 ± 5 µM and an apparent Vmax ≈ 14 pmol O2 uptake·mg−1·min−1 under the reported assay conditions. (hutcheson1985regulationof3indoleacetic pages 4-5)
A key regulatory feature is feedback inhibition by pathway intermediates: IAM is a strong competitive inhibitor (apparent Ki ≈ 7 µM, with ~50% inhibition at ~25 µM IAM at near-Km tryptophan), while IAA is substantially weaker (apparent inhibitory constant ~225–230 µM). (hutcheson1985regulationof3indoleacetic pages 4-5, hutcheson1985regulationof3indoleacetic pages 1-2)
Review synthesis notes that IAM-pathway genes in plant pathogens (e.g., P. savastanoi) can be plasmid-borne, highly expressed in planta, and that tryptophan can induce IAA-biosynthetic genes in some bacteria; however, regulation is not uniform across species/strains and is still considered incompletely understood. (duca2020indole3aceticacidbiosynthesis pages 8-9)
Kongjaroon et al. (RSC Chemical Biology, July 2024, https://doi.org/10.1039/d4cb00102h) improved thermostability of a Pseudomonas TMO while largely preserving kinetic function. The engineered variants TMO-PWS and TMO-PWSNR achieved Tm = 65 °C versus Tm = 48 °C for wild type (Table 3). (kongjaroon2024enhancementoftryptophan media 0c88a49e)
The same work reports steady-state kinetics (Figure 6A):
- WT: KM = 92.8 ± 5.9 µM, kcat = 8.07 ± 0.22 s−1
- PWS: KM = 117.2 ± 4.1 µM, kcat = 8.54 ± 0.21 s−1
- PWSNR: KM = 139.7 ± 9.2 µM, kcat = 8.81 ± 0.17 s−1 (kongjaroon2024enhancementoftryptophan media 0c88a49e)
Thermal inactivation half-lives highlight the magnitude of stabilization: at 50 °C, WT t1/2 = 2.2 min versus 183.8 min (PWS) and 199.6 min (PWSNR); at 60 °C, WT t1/2 = 0.4 min versus 14.2–14.7 min for engineered variants. (kongjaroon2024enhancementoftryptophan pages 7-8)
Relevance to functional annotation: these data reinforce that IaaM/TMO-family enzymes can be treated as modular biocatalysts (IAM production from tryptophan) and that protein stability is a key limitation for implementation, which 2024 work directly addresses. (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 7-8)
Khalil et al. (Microbiome, October 2024, https://doi.org/10.1186/s40168-024-01899-6) sequenced 14 diatom-associated marine bacterial isolates and reports that all strains possessed orthologues for one or more IAA biosynthesis routes, including the IAM pathway genes (IaaM, IaaH) listed in their pathway gene set/figure legend. (khalil2024widespreadproductionof pages 5-8)
They quantified extracellular IAA exudation and linked it to function: extracellular IAA was positively correlated with diatom growth (Pearson R = 0.73, p < 0.05), and experimental IAA supplementation at 10−7 µg/mL yielded a 2.5-fold increase in diatom cell numbers after 7 days relative to control conditions. (khalil2024widespreadproductionof pages 8-11)
Relevance to functional annotation: this positions bacterial IAA production (including IAM-pathway capacity) as an ecologically meaningful trait beyond classic plant pathogenesis, reinforcing modern interest in IaaM/TMO genes as determinants of cross-kingdom signaling. (khalil2024widespreadproductionof pages 5-8, khalil2024widespreadproductionof pages 8-11)
A 2023 perspective/review on engineering microbes to overproduce natural products for agricultural use (Synthetic and Systems Biotechnology, March 2023, https://doi.org/10.1016/j.synbio.2022.11.005) provides context for deploying engineered metabolic modules (including phytohormone pathways) as “agrochemicals,” supporting the broader trend that enzymes like IaaM/TMO can be repurposed in microbial chassis for agriculture-adjacent applications. (hutcheson1985regulationof3indoleacetic pages 2-2)
The engineered thermostable TMO variants (2024) are explicitly positioned as enabling “future studies” and improving soluble expression yield and stability while maintaining catalytic performance, which are core constraints for industrial enzyme deployment (e.g., IAM synthesis as an intermediate or as part of IAA-producing modules). (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 7-8)
The 2024 Microbiome study demonstrates a real-world ecological linkage between bacterial IAA exudation and enhanced diatom growth, suggesting that IAA biosynthetic potential (including IAM genes) can be a functional trait shaping microbial–eukaryote interactions in marine systems; exuded IAA reached isolate-specific maxima such as 2.7 ± 1 × 10−8 µg/cell in their dataset. (khalil2024widespreadproductionof pages 5-8)
If Q88D45 is indeed a canonical IaaM/TMO-family enzyme (as annotated), the best-supported biochemical function is:
- Enzymatic role: FAD-dependent monooxygenase catalyzing L-tryptophan + O2 → IAM + CO2 (EC 1.13.12.3). (hutcheson1985regulationof3indoleacetic pages 1-2, kongjaroon2024enhancementoftryptophan pages 1-2)
- Pathway placement: first step of the IAM pathway leading to IAA via an IAM hydrolase (IaaH). (duca2014indole3aceticacidin pages 8-9, duca2020indole3aceticacidbiosynthesis pages 8-9)
- Subcellular location: likely a soluble cytosolic enzyme, since canonical TMOs are purified from cell lysates and are not described as membrane-associated in primary biochemical work. (hutcheson1985regulationof3indoleacetic pages 2-3)
No retrieved KT2440-specific study demonstrated:
- IAM production from L-tryptophan by PP_4983/Q88D45 directly;
- the presence of a KT2440 iaaM–iaaH operon analogous to the plant-pathogen plasmid operon;
- a KT2440 phenotype attributable to PP_4983 in IAA production or plant interaction.
Moreover, the existence of divergent iaaM-like homologs in KT2440 reported in authoritative synthesis work (DavB/lysine degradation association) indicates that sequence similarity alone can mis-assign function in P. putida KT2440 without locus-level confirmation. (duca2014indole3aceticacidin pages 8-9)
Practical implication for annotation workflows: for Q88D45, reliable functional assignment should ideally be supported by (i) biochemical assay of L-tryptophan → IAM, (ii) genetic context analysis (presence/absence of a cognate IAM hydrolase and operon structure), and/or (iii) metabolomics demonstrating IAM/IAA intermediates linked to PP_4983 perturbation—none of which were available in the retrieved KT2440-focused corpus.
The table below consolidates quantitative kinetics, inhibition, and stability metrics from primary and recent work on canonical tryptophan 2-monooxygenases.
| Enzyme / source | Reaction | Cofactor | Kinetic parameters | Inhibition / feedback | Substrate scope | Thermostability / stability | Evidence |
|---|---|---|---|---|---|---|---|
| Tryptophan 2-monooxygenase (IaaM/TMO), Pseudomonas syringae pv. savastanoi | Oxidative decarboxylation of L-tryptophan to indole-3-acetamide (IAM) with O2 uptake; first step of the IAM→IAA pathway (hutcheson1985regulationof3indoleacetic pages 1-2) | One FAD per ~62 kDa subunit; flavoprotein (hutcheson1985regulationof3indoleacetic pages 1-2, hutcheson1985regulationof3indoleacetic pages 4-5) | Apparent Km(L-Trp) ≈ 50 ± 5 µM; apparent Vmax ≈ 14 pmol O2 uptake·mg^-1·min^-1 under reported assay conditions (hutcheson1985regulationof3indoleacetic pages 4-5) | IAM is a strong competitive feedback inhibitor: apparent Ki ≈ 7 µM; ~50% inhibition at ~25 µM IAM. IAA is a weaker inhibitor: apparent Ki / inhibitory constant around 225–230 µM; ~50% inhibition at ~230 µM IAA (hutcheson1985regulationof3indoleacetic pages 4-5, hutcheson1985regulationof3indoleacetic pages 1-2) | Accepts several substituted tryptophans including 4-, 5-, 6-, 7-methyltryptophan, 7-azatryptophan, 5-fluorotryptophan, and 5-hydroxytryptophan; does not accept D-tryptophan or α-methyltryptophan (hutcheson1985regulationof3indoleacetic pages 4-5) | No Tm reported in classic study; enzyme noted to be unstable during purification, but stabilized in alkaline buffer and by IAM during purification (hutcheson1985regulationof3indoleacetic pages 2-3) | (hutcheson1985regulationof3indoleacetic pages 4-5, hutcheson1985regulationof3indoleacetic pages 1-2, hutcheson1985regulationof3indoleacetic pages 2-3) |
| Tryptophan 2-monooxygenase (IaaM/TMO), Pseudomonas savastanoi / P. savastanoi pv. savastanoi | L-tryptophan + O2 → IAM + CO2; first committed step in IAM-pathway IAA biosynthesis (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 2-4) | FAD-bound flavoenzyme; FAD reduced by substrate and reoxidized by O2 (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 2-4, hutcheson1985regulationof3indoleacetic pages 4-5) | Classic WT kinetic benchmark from the same enzyme family/source lineage: Km(L-Trp) ~50 µM in 1985 purification study (hutcheson1985regulationof3indoleacetic pages 1-2, hutcheson1985regulationof3indoleacetic pages 4-5) | TMO activity inhibited by IAM and IAA; feedback regulation discussed in review of IAM pathway regulation (duca2020indole3aceticacidbiosynthesis pages 8-9) | Can accept other aromatic amino acids in some contexts; engineered biocatalysis literature notes phenylacetamide production from other aromatic amino acids (kongjaroon2024enhancementoftryptophan pages 1-2) | Not quantitatively summarized for native enzyme in retrieved modern review excerpts (duca2020indole3aceticacidbiosynthesis pages 8-9) | (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 2-4, hutcheson1985regulationof3indoleacetic pages 4-5, duca2020indole3aceticacidbiosynthesis pages 8-9) |
| TMO-WT, engineered-variant study source: Pseudomonas savastanoi (recombinant enzyme in 2024 thermostability study) | L-tryptophan + O2 → IAM + CO2; IAM subsequently converted to IAA by IaaH (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 2-4) | Enzyme-bound FAD monitored at 466 nm; FAD used in MD and biochemical analyses (kongjaroon2024enhancementoftryptophan pages 11-12, kongjaroon2024enhancementoftryptophan pages 9-10) | Km = 92.8 ± 5.9 µM; kcat = 8.07 ± 0.22 s^-1 (kongjaroon2024enhancementoftryptophan media 0c88a49e) | No inhibition constants reported in 2024 engineering paper excerpt (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan media 0c88a49e) | IAM formation quantified by HPLC-MS; substrate tested was L-tryptophan in steady-state assays (kongjaroon2024enhancementoftryptophan pages 11-12, kongjaroon2024enhancementoftryptophan media 0c88a49e) | Tm = 48 °C; t1/2 at 50 °C = 2.2 min; t1/2 at 60 °C = 0.4 min (kongjaroon2024enhancementoftryptophan pages 7-8, kongjaroon2024enhancementoftryptophan media 0c88a49e) | (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 7-8, kongjaroon2024enhancementoftryptophan pages 11-12, kongjaroon2024enhancementoftryptophan pages 9-10, kongjaroon2024enhancementoftryptophan media 0c88a49e) |
| TMO-PWS, engineered thermostable variant | Same as WT: L-tryptophan + O2 → IAM + CO2 (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 2-4) | FAD-bound enzyme (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 11-12) | Km = 117.2 ± 4.1 µM; kcat = 8.54 ± 0.21 s^-1 (kongjaroon2024enhancementoftryptophan media 0c88a49e) | No inhibition constants reported (kongjaroon2024enhancementoftryptophan media 0c88a49e) | Retained IAM-producing activity with catalytic properties described as similar to WT (kongjaroon2024enhancementoftryptophan pages 9-10, kongjaroon2024enhancementoftryptophan media 0c88a49e) | Tm = 65 °C; t1/2 at 50 °C = 183.8 min; t1/2 at 60 °C = 14.2 min; >85% residual activity after 1 h at 50 °C (kongjaroon2024enhancementoftryptophan pages 7-8, kongjaroon2024enhancementoftryptophan media 0c88a49e) | (kongjaroon2024enhancementoftryptophan pages 7-8, kongjaroon2024enhancementoftryptophan pages 9-10, kongjaroon2024enhancementoftryptophan media 0c88a49e) |
| TMO-PWSNR, engineered thermostable variant | Same as WT: L-tryptophan + O2 → IAM + CO2 (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 2-4) | FAD-bound enzyme (kongjaroon2024enhancementoftryptophan pages 1-2, kongjaroon2024enhancementoftryptophan pages 11-12) | Km = 139.7 ± 9.2 µM; kcat = 8.81 ± 0.17 s^-1 (kongjaroon2024enhancementoftryptophan media 0c88a49e) | No inhibition constants reported (kongjaroon2024enhancementoftryptophan media 0c88a49e) | Retained IAM-producing activity with catalytic properties described as similar to WT (kongjaroon2024enhancementoftryptophan pages 9-10, kongjaroon2024enhancementoftryptophan media 0c88a49e) | Tm = 65 °C; t1/2 at 50 °C = 199.6 min; t1/2 at 60 °C = 14.7 min; >85% residual activity after 1 h at 50 °C (kongjaroon2024enhancementoftryptophan pages 7-8, kongjaroon2024enhancementoftryptophan media 0c88a49e) | (kongjaroon2024enhancementoftryptophan pages 7-8, kongjaroon2024enhancementoftryptophan pages 9-10, kongjaroon2024enhancementoftryptophan media 0c88a49e) |
Table: This table compiles key biochemical, kinetic, inhibition, substrate-scope, and thermostability data for bacterial tryptophan 2-monooxygenase/IaaM from the retrieved evidence. It is useful for comparing canonical plant-pathogen TMO properties with the 2024 engineered thermostable variants.
Key quantitative evidence from 2024 enzyme engineering is shown directly in the cropped figure/table images (KM/kcat and Tm). (kongjaroon2024enhancementoftryptophan media 0c88a49e, kongjaroon2024enhancementoftryptophan media 382043ec)
References
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(yamada1985nucleotidesequencesof pages 3-4): Tetsuji Yamada, Curtis J. Palm, Bob Brooks, and Tsune Kosuge. Nucleotide sequences of the pseudomonas savastanoi indoleacetic acid genes show homology with agrobacterium tumefaciens t-dna. Proceedings of the National Academy of Sciences of the United States of America, 82 19:6522-6, Oct 1985. URL: https://doi.org/10.1073/pnas.82.19.6522, doi:10.1073/pnas.82.19.6522. This article has 288 citations and is from a highest quality peer-reviewed journal.
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(kongjaroon2024enhancementoftryptophan pages 1-2): Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat, and Pimchai Chaiyen. Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation. RSC Chemical Biology, 5:989-1001, Jul 2024. URL: https://doi.org/10.1039/d4cb00102h, doi:10.1039/d4cb00102h. This article has 6 citations and is from a peer-reviewed journal.
(kongjaroon2024enhancementoftryptophan pages 2-4): Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat, and Pimchai Chaiyen. Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation. RSC Chemical Biology, 5:989-1001, Jul 2024. URL: https://doi.org/10.1039/d4cb00102h, doi:10.1039/d4cb00102h. This article has 6 citations and is from a peer-reviewed journal.
(kongjaroon2024enhancementoftryptophan pages 9-10): Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat, and Pimchai Chaiyen. Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation. RSC Chemical Biology, 5:989-1001, Jul 2024. URL: https://doi.org/10.1039/d4cb00102h, doi:10.1039/d4cb00102h. This article has 6 citations and is from a peer-reviewed journal.
(kongjaroon2024enhancementoftryptophan pages 11-12): Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat, and Pimchai Chaiyen. Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation. RSC Chemical Biology, 5:989-1001, Jul 2024. URL: https://doi.org/10.1039/d4cb00102h, doi:10.1039/d4cb00102h. This article has 6 citations and is from a peer-reviewed journal.
(kongjaroon2024enhancementoftryptophan media 0c88a49e): Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat, and Pimchai Chaiyen. Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation. RSC Chemical Biology, 5:989-1001, Jul 2024. URL: https://doi.org/10.1039/d4cb00102h, doi:10.1039/d4cb00102h. This article has 6 citations and is from a peer-reviewed journal.
(kongjaroon2024enhancementoftryptophan pages 7-8): Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat, and Pimchai Chaiyen. Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation. RSC Chemical Biology, 5:989-1001, Jul 2024. URL: https://doi.org/10.1039/d4cb00102h, doi:10.1039/d4cb00102h. This article has 6 citations and is from a peer-reviewed journal.
(khalil2024widespreadproductionof pages 5-8): Abeeha Khalil, Anna R. Bramucci, Amaranta Focardi, Nine Le Reun, Nathan L. R. Willams, Unnikrishnan Kuzhiumparambil, Jean-Baptiste Raina, and Justin R. Seymour. Widespread production of plant growth-promoting hormones among marine bacteria and their impacts on the growth of a marine diatom. Microbiome, Oct 2024. URL: https://doi.org/10.1186/s40168-024-01899-6, doi:10.1186/s40168-024-01899-6. This article has 11 citations and is from a highest quality peer-reviewed journal.
(khalil2024widespreadproductionof pages 8-11): Abeeha Khalil, Anna R. Bramucci, Amaranta Focardi, Nine Le Reun, Nathan L. R. Willams, Unnikrishnan Kuzhiumparambil, Jean-Baptiste Raina, and Justin R. Seymour. Widespread production of plant growth-promoting hormones among marine bacteria and their impacts on the growth of a marine diatom. Microbiome, Oct 2024. URL: https://doi.org/10.1186/s40168-024-01899-6, doi:10.1186/s40168-024-01899-6. This article has 11 citations and is from a highest quality peer-reviewed journal.
(hutcheson1985regulationof3indoleacetic pages 2-2): S W Hutcheson and T Kosuge. Regulation of 3-indoleacetic acid production in pseudomonas syringae pv. savastanoi. purification and properties of tryptophan 2-monooxygenase. The Journal of biological chemistry, 260 10:6281-7, May 1985. URL: https://doi.org/10.1016/s0021-9258(18)88968-2, doi:10.1016/s0021-9258(18)88968-2. This article has 95 citations.
(hutcheson1985regulationof3indoleacetic pages 2-3): S W Hutcheson and T Kosuge. Regulation of 3-indoleacetic acid production in pseudomonas syringae pv. savastanoi. purification and properties of tryptophan 2-monooxygenase. The Journal of biological chemistry, 260 10:6281-7, May 1985. URL: https://doi.org/10.1016/s0021-9258(18)88968-2, doi:10.1016/s0021-9258(18)88968-2. This article has 95 citations.
(kongjaroon2024enhancementoftryptophan media 382043ec): Sirus Kongjaroon, Narin Lawan, Duangthip Trisrivirat, and Pimchai Chaiyen. Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation. RSC Chemical Biology, 5:989-1001, Jul 2024. URL: https://doi.org/10.1039/d4cb00102h, doi:10.1039/d4cb00102h. This article has 6 citations and is from a peer-reviewed journal.
iaaM does not resolve as an exact UniProt gene symbol in PSEPK. The candidate reviewed here is Q88D45 / PP_4983 because its UniProt record carries the automated tryptophan 2-monooxygenase and auxin-pathway assignment. [UniProt Q88D45 "DE RecName: Full=Tryptophan 2-monooxygenase"; "GN OrderedLocusNames=PP_4983"]PE 3: Inferred from homology, Belongs to the tryptophan 2-monooxygenase family, and PATHWAY: Plant hormone metabolism; auxin biosynthesis. [UniProt Q88D45 "PE 3: Inferred from homology;"; "Belongs to the tryptophan 2-monooxygenase family."; "PATHWAY: Plant hormone metabolism; auxin biosynthesis."]The YAML description field was revised to keep it as a standalone biological summary. Project-specific curation framing moved here instead.
id: Q88D45
gene_symbol: PP_4983
aliases:
- iaaM
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:160488
label: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950
/ KT2440)
description: >-
PP_4983 is an exported FAD-dependent amine oxidase family protein in Pseudomonas putida KT2440. The
locus is often discussed under the alias iaaM because UniProt/GO automation assigns tryptophan
2-monooxygenase activity and auxin biosynthesis to it, but the specific iaaM interpretation is weak.
The same locus is annotated by KEGG as a putative amine oxidase / monoamine oxidase (K00274, EC
1.4.3.4), and KT2440 already contains distinct amino oxidase family members including davB/PP_0383.
The best current synthesis is monoamine-oxidase-like oxidoreductase activity with unresolved
physiological substrate, rather than a confirmed indole-3-acetamide auxin-biosynthetic enzyme.
existing_annotations:
- term:
id: GO:0016491
label: oxidoreductase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: PP_4983 is clearly an oxidoreductase-family protein, but this parent
term is too broad to be useful. Both UniProt and KEGG place the protein within
flavin-dependent amine oxidation chemistry, so a more specific amine-oxidase-like
molecular function is preferable.
action: MARK_AS_OVER_ANNOTATED
reason: The broad oxidoreductase root term adds little information once the locus
is interpreted as a monoamine-oxidase-like flavoprotein. It is directionally
correct but not an informative core annotation.
supported_by:
- reference_id: file:PSEPK/iaaM/iaaM-notes.md
supporting_text: The safest current synthesis is amine oxidase / monoamine oxidase-like
flavoprotein rather than confirmed tryptophan 2-monooxygenase in auxin biosynthesis.
- term:
id: GO:0050361
label: tryptophan 2-monooxygenase activity
evidence_type: IEA
original_reference_id: GO_REF:0000003
review:
summary: This specific iaaM-style annotation is not well supported for PP_4983.
The UniProt assignment is homology-based only, whereas KEGG classifies the same
locus as a putative amine oxidase / monoamine oxidase and not as a dedicated
indole-3-acetamide auxin enzyme. In the same genome, davB/PP_0383 is already
assigned a distinct amino-oxidase chemistry, reinforcing that automatic family
transfer is likely overspecific here.
action: MODIFY
reason: The protein is probably an amine oxidase, but the specific substrate assignment
to tryptophan-to-indole-3-acetamide conversion is weaker than the broader monoamine
oxidase interpretation from KEGG. Until biochemical data establish IAM formation
in KT2440, the safer molecular function is monoamine oxidase activity.
proposed_replacement_terms:
- id: GO:0097621
label: monoamine oxidase activity
supported_by:
- reference_id: file:PSEPK/iaaM/iaaM-uniprot.txt
supporting_text: Belongs to the tryptophan 2-monooxygenase family.
- reference_id: file:PSEPK/iaaM/iaaM-uniprot.txt
supporting_text: 'PE 3: Inferred from homology;'
- reference_id: file:PSEPK/iaaM/iaaM-notes.md
supporting_text: KEGG contradicts the specific iaaM call for the same locus
and instead labels PP_4983 as putative amine oxidase with orthology K00274
monoamine oxidase [EC:1.4.3.4].
core_functions:
- molecular_function:
id: GO:0097621
label: monoamine oxidase activity
description: 'Best current synthesis: PP_4983 is most likely an exported FAD-dependent
monoamine oxidase / amine oxidase family protein. The exact in vivo substrate
remains unresolved, and the available evidence does not justify confident assignment
to indole-3-acetamide auxin biosynthesis in KT2440.'
supported_by:
- reference_id: file:PSEPK/iaaM/iaaM-notes.md
supporting_text: The safest current synthesis is amine oxidase / monoamine oxidase-like
flavoprotein rather than confirmed tryptophan 2-monooxygenase in auxin biosynthesis.
suggested_questions:
- question: Is PP_4983 able to convert L-tryptophan to indole-3-acetamide in vitro
or in vivo, or is its physiological substrate a different monoamine?
- question: Does KT2440 encode a bona fide iaaH-like amidase that pairs with PP_4983
in an indole-3-acetamide pathway?
suggested_experiments:
- hypothesis: PP_4983 is a monoamine oxidase-like flavoprotein rather than a dedicated
iaaM tryptophan 2-monooxygenase.
description: Purify PP_4983 and test FAD-dependent oxidation of L-tryptophan, methylamine,
phenethylamine, tyramine, and related amines by LC-MS or targeted product assays.
Compare turnover against authentic indole-3-acetamide production.
experiment_type: biochemical enzyme assay
- hypothesis: If PP_4983 participates in IAM/IAA biosynthesis, deleting the locus
should reduce indole-3-acetamide or indole-3-acetic acid accumulation from tryptophan.
description: Construct a PP_4983 deletion strain and quantify indole-3-acetamide
and indole-3-acetic acid in wild type and mutant cultures grown with supplemental
tryptophan.
experiment_type: genetics plus metabolomics
- hypothesis: PP_4983 and the other KT2440 amine oxidase candidates have distinct
substrate scopes.
description: Compare PP_4983, PP_4155, and davB mutant growth and transcriptional
responses across candidate amine and amino-acid substrates to separate auxin-pathway
and general amine-catabolic roles.
experiment_type: comparative genetics and physiology
references:
- id: GO_REF:0000003
title: Gene Ontology annotation based on Enzyme Commission mapping
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: file:PSEPK/iaaM/iaaM-uniprot.txt
title: UniProt text record for PP_4983 / Q88D45
findings:
- statement: UniProt assigns PP_4983 to the tryptophan 2-monooxygenase family by
homology.
supporting_text: Belongs to the tryptophan 2-monooxygenase family.
- statement: The auxin pathway assignment in UniProt is homology-based rather than
experimental.
supporting_text: 'PE 3: Inferred from homology;'
- id: file:PSEPK/iaaM/iaaM-notes.md
title: Notes on the PP_4983 iaaM candidate review
findings:
- statement: KEGG assigns PP_4983 as putative amine oxidase / monoamine oxidase
rather than iaaM.
supporting_text: KEGG contradicts the specific iaaM call for the same locus and
instead labels PP_4983 as putative amine oxidase with orthology K00274 monoamine
oxidase [EC:1.4.3.4].
- statement: KT2440 contains additional amino oxidase family members, including
davB/PP_0383.
supporting_text: KT2440 already contains another amino oxidase family member,
PP_4155, also labeled putative amine oxidase, and a distinct lysine-pathway
enzyme davB / PP_0383 assigned K00468 lysine 2-monooxygenase [EC:1.13.12.2].
- id: PMID:12534463
title: Complete genome sequence and comparative analysis of the metabolically versatile
Pseudomonas putida KT2440
full_text_unavailable: true
findings:
- statement: Establishes the PP_4983 locus as part of the KT2440 genome sequence.