GO Term	Evidence	Action	Reason
GO:0003730 mRNA 3'-UTR binding	IEA GO_REF:0000117	MARK AS OVER ANNOTATED	Summary: ARBA machine-learning prediction of an RNA-binding moonlighting function, not supported by organism-specific evidence. Reason: This electronic ARBA prediction transfers an apo-aconitase moonlighting function demonstrated in other bacteria. No transcript-binding or post-transcriptional-regulation evidence was found for Q88KF1 in P. putida, so the term should not be treated as established for this target. Supporting Evidence: file:PSEPK/acnB/acnB-deep-research-openscientist.md Whether P. putida AcnB binds specific transcripts, and which ones, has not been established
GO:0003994 aconitate hydratase activity	IEA GO_REF:0000120	ACCEPT	Summary: Core catalytic activity of AcnB, well supported by family/EC assignment and organism-specific pathway placement. Reason: Q88KF1 belongs to the aconitase/IPM-isomerase family, and UniProt assigns the citrate-isocitrate reaction and EC 4.2.1.3. This is a core molecular function. Supporting Evidence: file:PSEPK/acnB/acnB-uniprot.txt Reaction=citrate = D-threo-isocitrate;
GO:0005829 cytosol	IEA GO_REF:0000120	ACCEPT	Summary: AcnB is a soluble enzyme of the TCA/methylcitrate central metabolic network, best inferred to act in the cytosol. Reason: AcnB is a soluble central-metabolic enzyme with no secretion or membrane role. The InterPro-derived cytosol assignment is consistent with its substrates and conserved bacterial aconitase function.
GO:0006099 tricarboxylic acid cycle	IEA GO_REF:0000120	ACCEPT	Summary: AcnB catalyzes the aconitase step of the TCA cycle; a core biological process for this enzyme. Reason: The citrate-isocitrate aconitase reaction is a defining TCA-cycle step, and UniProt explicitly places Q88KF1 in this pathway. Supporting Evidence: file:PSEPK/acnB/acnB-uniprot.txt PATHWAY: Carbohydrate metabolism; tricarboxylic acid cycle; isocitrate
GO:0047456 2-methylisocitrate dehydratase activity	IEA GO_REF:0000120	ACCEPT	Summary: Methylcitrate-cycle dehydratase activity (EC 4.2.1.99) of bifunctional AcnB, supported by organism-specific pathway genetics. Reason: UniProt assigns EC 4.2.1.99 to Q88KF1. Biochemical reconstitution with the Salmonella ortholog shows that AcnB catalyzes conversion of 2-methyl-cis-aconitate to 2-methylisocitrate. Supporting Evidence: PMID:11294638 homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate file:PSEPK/acnB/acnB-deep-research-openscientist.md AcnB catalyzes the hydration of *2-methyl-cis-aconitate to 2-methylisocitrate* (EC 4.2.1.99)
GO:0051539 4 iron, 4 sulfur cluster binding	IEA GO_REF:0000120	ACCEPT	Summary: AcnB requires a catalytic [4Fe-4S] cluster, an essential cofactor of aconitase-family enzymes. Reason: A catalytic [4Fe-4S] cluster is a defining mechanistic feature of aconitases, and UniProt assigns one cluster per Q88KF1 subunit. Supporting Evidence: file:PSEPK/acnB/acnB-uniprot.txt Name=[4Fe-4S] cluster;
GO:0019543 propionate catabolic process	IC file:PSEPK/acnB/acnB-uniprot.txt	NEW	Summary: Process annotation for AcnB's role in propionate catabolism. Reason: UniProt places Q88KF1 in propanoate degradation, and the accepted 2-methylisocitrate dehydratase activity is the aconitase step of the methylcitrate cycle. Supporting Evidence: file:PSEPK/acnB/acnB-uniprot.txt PATHWAY: Organic acid metabolism; propanoate degradation.

Deep Research

Falcon

(acnB-deep-research-falcon.md)

Research Report: Functional Annotation of **acnB** (UniProt Q88KF1; locus **PP_2339**) in *Pseudomonas putida* KT2440 Falcon Edison Scientific Literature 24 citations 2 artifacts 2026-06-11T16:46:02.544891

Edison artifact artifact-00 (text/markdown)

## Context ID: pqac-00000011 The methylcitrate cycle (MCC), which is essential for propionate and odd-chain fatty acid catabolism, is illustrated in the schemat (image/png)

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.

Research Report: Functional Annotation of acnB (UniProt Q88KF1; locus PP_2339) in Pseudomonas putida KT2440

0. Target verification (critical disambiguation)

The gene symbol acnB is used broadly across bacteria, but in Pseudomonas putida KT2440 the locus PP_2339 is explicitly annotated in KT2440-focused studies as acnB, encoding a bifunctional 2-methylcitrate dehydratase/aconitase hydratase B—consistent with the UniProt-provided identity for Q88KF1. (thompson2020fattyacidand pages 5-7, thompson2020fattyacidand pages 2-5, thompson2020functionalanalysisof pages 8-12)

1. Key concepts and current definitions

1.1 Aconitase / aconitate hydratase (EC 4.2.1.3)

“Aconitase” (aconitate hydratase) is a central-carbon enzyme classically positioned in the tricarboxylic acid (TCA) cycle, catalyzing the reversible isomerization of citrate to isocitrate via cis-aconitate. In bacteria, aconitase activity is typically carried by isoenzymes (often AcnA and AcnB). A key mechanistic feature is that enzymatic activity requires an intact [4Fe–4S] iron–sulfur cluster; disruption (e.g., oxidative damage or low iron) yields an inactive form. (crack2018redoxsensingiron–sulfurcluster pages 23-25, austin2015aconitasefunctionsas pages 1-2)

1.2 Methylcitrate cycle (MCC) and “bifunctional” AcnB

In P. putida KT2440, acnB (PP_2339) is discussed as part of the methylcitrate cycle (MCC), a pathway that assimilates/detoxifies propionyl-CoA (commonly produced during β-oxidation of odd-chain fatty acids) into central metabolites (succinate and pyruvate), which feed into the broader TCA/central metabolism network. In this KT2440 context, AcnB is annotated as bifunctional: it can contribute to both classical aconitase chemistry and MCC-associated dehydratase/hydratase steps involving methylcitrate intermediates. (thompson2020fattyacidand pages 5-7, thompson2020fattyacidand pages 2-5, thompson2020fattyacidand media b8c40d82)

1.3 Apo-aconitase “moonlighting” as an RNA-binding protein (general bacterial concept)

Authoritative mechanistic literature shows that when aconitase loses/disrupts its [4Fe–4S] cluster (forming apo-aconitase), the protein can adopt RNA-binding post-transcriptional regulatory roles (e.g., binding specific mRNAs and altering stability/translation). This “switch” between enzymatic and RNA-binding roles is supported in multiple bacteria; direct experimental evidence exists in Helicobacter pylori and conceptually in E. coli reviewed literature, while it has not yet been demonstrated for P. putida KT2440 PP_2339 in the retrieved corpus. (crack2018redoxsensingiron–sulfurcluster pages 23-25, austin2015aconitasefunctionsas pages 1-2)

2. Functional role of P. putida KT2440 AcnB (PP_2339; Q88KF1)

2.1 Primary biochemical function: central-carbon dehydratase/isomerase chemistry

Best-supported organism-specific functional claim: In P. putida KT2440, acnB (PP_2339) functions in the methylcitrate cycle and is annotated as bifunctional 2-methylcitrate dehydratase/aconitate hydratase B. (thompson2020fattyacidand pages 5-7, thompson2020functionalanalysisof pages 8-12)

Substrate specificity (evidence status):
* KT2440-specific full-text evidence in the retrieved set provides pathway-level functional assignment (MCC involvement) and “bifunctional” annotation but does not provide detailed kinetic parameters (Km/kcat) or explicit chemical equations for PP_2339. (thompson2020fattyacidand pages 5-7, thompson2020functionalanalysisof pages 8-12)
* Mechanistic enzyme/cofactor principles (Fe–S dependence) come from broader aconitase literature rather than KT2440-specific biochemical assays. (crack2018redoxsensingiron–sulfurcluster pages 23-25, austin2015aconitasefunctionsas pages 1-2)

2.2 Pathway context: methylcitrate cycle is required for odd-chain fatty acid utilization

A key experimental systems-genetics result in KT2440 is that the methylcitrate cycle is essential for growth on odd-chain fatty acids, specifically noted as propionate (C3), valerate (C5), heptanoate (C7), and nonanoate (C9). The same work places PP_2339 (acnB) in the MCC and argues it likely carries key MCC dehydratase/hydratase activity, especially because a canonical MCC dehydratase gene (prpD/PP_2338) did not show a fitness defect. (thompson2020fattyacidand pages 5-7, thompson2020fattyacidand media b8c40d82)

Visual evidence (pathway placement): Figure 2 in Thompson et al. (2020) depicts the MCC converting propionyl-CoA into succinate and pyruvate and presents a gene-fitness heatmap for odd-chain fatty acid growth conditions. (thompson2020fattyacidand media b8c40d82, thompson2020fattyacidand media 3b2d456f)

2.3 Cellular localization

No KT2440-specific microscopy/fractionation evidence for PP_2339 localization was retrieved. However, the enzymes and pathways described (TCA and MCC) are presented as part of the soluble central metabolic network; thus, AcnB is best inferred to function primarily in the cytosol. (thompson2020fattyacidand pages 5-7, molina2019pseudomonasputidakt2440 pages 8-9)

3. Phenotypes, essentiality, and regulation in P. putida KT2440

3.1 Essentiality / genetic constraint

Random barcode transposon sequencing (RB-TnSeq) analyses in KT2440 report no mapped transposon insertions in PP_2339 (acnB). The authors interpret this absence as suggesting that PP_2339 was essential during construction of the RB–TnSeq library (i.e., disruption may be lethal or severely deleterious under the baseline conditions used to build/maintain the library). (thompson2020fattyacidand pages 5-7, thompson2020functionalanalysisof pages 8-12)

This is an inference from insertion absence rather than a classical targeted knockout with complementation; nonetheless, in large mutant libraries, lack of insertions is a common hallmark of essentiality or strong growth constraint. (thompson2020fattyacidand pages 5-7)

3.2 Expression/proteomic behavior in growth phases

Quantitative proteomic profiling of KT2440 growth in complete medium indicates that aconitase isoforms can vary by growth phase; in the cited excerpt, AcnB abundance was reported as similar in early and mid exponential phases, while some other isoforms (e.g., AcnA) decreased. This supports AcnB as a constitutive component of central metabolism across growth stages in this setting. (molina2019pseudomonasputidakt2440 pages 8-9)

3.3 Oxidative stress / iron limitation considerations (evidence boundaries)

KT2440-specific evidence directly tying PP_2339 regulation to oxidative stress/iron limitation was not retrieved.

However, authoritative mechanistic work in bacteria shows:
* Aconitase enzymatic activity depends on an intact [4Fe–4S] cluster; oxidation (high oxygen/oxidants) or iron scarcity can inactivate aconitase via cluster disruption. (crack2018redoxsensingiron–sulfurcluster pages 23-25, austin2015aconitasefunctionsas pages 1-2)
* Under these conditions, cluster-free aconitase can switch to RNA-binding regulatory roles in several bacteria (e.g., H. pylori AcnB binding multiple transcripts under O2 stress). (austin2015aconitasefunctionsas pages 1-2)

For KT2440 functional annotation, these findings motivate a plausible hypothesis that PP_2339 may also be stress-sensitive and potentially moonlighting, but this should be labeled as family-supported inference pending KT2440-specific experiments.

4. Recent developments (prioritizing 2023–2024)

4.1 2023: genome-scale functional genetics resources for KT2440

A 2023 KT2440 genome-mining/functional-genomics perspective explicitly identifies PP_2339 (acnB) as a bifunctional 2-methylcitrate dehydratase/aconitase hydratase B and notes the absence of mapped insertions consistent with an essential gene in an RB-TnSeq library context. (thompson2020functionalanalysisof pages 8-12)

4.2 2024: applied central-metabolism rewiring for aromatic compound degradation

A 2024 Microbial Biotechnology study demonstrates that inactivating pyruvate dehydrogenase (PDH) in KT2440 relieves carbon catabolite repression and improves the strain’s ability to degrade aromatic compounds (many of which funnel to acetyl-CoA and enter central metabolism via the TCA cycle). This underscores the importance of TCA-connected nodes (pyruvate → acetyl-CoA entry; downstream TCA function including aconitase steps) in real-world biotransformation/bioremediation performance. (moreno2024inactivationofpseudomonas pages 1-2)

4.3 2024: authoritative demonstration of iron-stress aconitase moonlighting (cross-species mechanistic advance)

A 2024 Nucleic Acids Research study (in Staphylococcus aureus) provides high-quality evidence that aconitase can be tightly regulated under iron deficiency by an sRNA-driven circuit and that aconitase itself can exert moonlighting RNA-binding activity that downregulates its own expression, illustrating conserved regulatory logic around Fe–S-dependent central metabolism. While not in P. putida, this strengthens the mechanistic precedent for Fe–S enzymes acting as metabolic/stress sensors. (barrault2024staphylococcalaconitaseexpression pages 1-3, barrault2024staphylococcalaconitaseexpression pages 12-14)

5. Current applications and real-world implementations

5.1 Bioremediation/biotransformation requiring robust central metabolism

KT2440 is widely used as a chassis for degradation of aromatic compounds; a 2024 study shows that rewiring central carbon entry (PDH-null) improves degradation of aromatic compounds even in the presence of preferred substrates, emphasizing industrial relevance of central metabolism and TCA-cycle throughput. Although AcnB is not singled out, aconitase activity is a core TCA step, so AcnB’s function is part of the metabolic backbone enabling such applications. (moreno2024inactivationofpseudomonas pages 1-2)

5.2 Odd-chain fatty acid utilization and propionyl-CoA detoxification

The methylcitrate cycle (in which PP_2339/acnB is placed) is essential for growth on odd-chain fatty acids, implying that AcnB supports utilization of feedstocks that yield propionyl-CoA. This is directly relevant to metabolic engineering contexts where fatty acids or complex substrates produce odd-chain intermediates that would otherwise be toxic or poorly assimilated. (thompson2020fattyacidand pages 5-7)

6. Relevant statistics and data (from available sources)

Essentiality inference: PP_2339 (acnB) lacks RB-TnSeq insertions; authors interpret this as essential during library construction (qualitative essentiality evidence). (thompson2020fattyacidand pages 5-7, thompson2020functionalanalysisof pages 8-12)
Substrate class requirement: The methylcitrate cycle (placing acnB as a key component) is described as absolutely required for growth on propionate (C3), valerate (C5), heptanoate (C7), nonanoate (C9). (thompson2020fattyacidand pages 5-7)
Proteomics trend: AcnB abundance is reported as similar between early and mid exponential phase in KT2440 growth in complete medium (qualitative expression statistic). (molina2019pseudomonasputidakt2440 pages 8-9)

Data not recovered in accessible full text: Numerical kinetic constants for KT2440 AcnB (Km, kcat), explicit quantitative transposon fitness values for acnB itself (since it lacks insertions), and KT2440-specific iron/oxygen stress regulation experiments.

7. Expert analysis and synthesis

7.1 Most defensible functional annotation for Q88KF1 in KT2440

The highest-confidence KT2440-specific annotation is that AcnB (PP_2339; Q88KF1) is a cytosolic, Fe–S-dependent aconitase-family enzyme that plays a critical role in propionyl-CoA assimilation via the methylcitrate cycle, supporting growth on odd-chain fatty acids and likely providing the principal methylaconitate/2-methylcitrate isomerization/dehydration capacity in this pathway. (thompson2020fattyacidand pages 5-7, thompson2020fattyacidand media b8c40d82)

7.2 Why “bifunctional” matters in P. putida

The KT2440 genetics strongly suggest functional redundancy/partitioning among paralogs: despite the presence of prpD (PP_2338), no fitness defect was observed for that gene on odd-chain fatty acids, and the authors specifically infer that PP_2339 likely performs the relevant MCC dehydratase function. This is a concrete example of how sequence-based annotation alone can mislead without functional genetics. (thompson2020fattyacidand pages 5-7)

7.3 Stress biology: what can be inferred vs what is proven

Because AcnB-type aconitases rely on [4Fe–4S] clusters that are susceptible to oxidative disruption and iron limitation—and because apo-forms can bind RNA in other bacteria—KT2440 AcnB is plausibly both a metabolic catalyst and a potential stress-responsive node. Yet, this report should not claim KT2440 RNA-binding moonlighting without direct evidence; instead, it is best presented as a mechanistically grounded hypothesis for future validation in KT2440. (crack2018redoxsensingiron–sulfurcluster pages 23-25, austin2015aconitasefunctionsas pages 1-2)

Embedded summary table

Aspect	Key points	Best supporting sources
Identity	Target is acnB / PP_2339 in Pseudomonas putida KT2440; KT2440-focused studies annotate it as a bifunctional 2-methylcitrate dehydratase/aconitase hydratase B, matching UniProt Q88KF1.	(thompson2020fattyacidand pages 5-7, thompson2020fattyacidand pages 2-5, thompson2020functionalanalysisof pages 8-12)
Enzymatic activities	Evidence supports dual activity as aconitate hydratase (aconitase) in central metabolism and 2-methylcitrate/2-methylisocitrate dehydratase-related activity in the methylcitrate cycle; literature places AcnB at the rehydration/dehydration steps around methylaconitate/2-methylisocitrate.	(thompson2020fattyacidand pages 5-7, thompson2020fattyacidand pages 2-5, thompson2020functionalanalysisof pages 8-12, thompson2020fattyacidand media b8c40d82)
Pathway roles	AcnB links the TCA cycle and the methylcitrate cycle (MCC) used for assimilation/detoxification of propionyl-CoA derived from odd-chain fatty acids; MCC yields succinate and pyruvate for central metabolism.	(thompson2020fattyacidand pages 5-7, thompson2020fattyacidand pages 2-5, molina2019pseudomonasputidakt2440 pages 8-9, thompson2020fattyacidand media b8c40d82)
Cofactor & sensitivity	As an aconitase-family enzyme, AcnB is expected to use a [4Fe-4S] cluster; broader aconitase literature indicates this cofactor is required for catalysis and is sensitive to oxidation/iron limitation, with AcnB often less stable than AcnA under oxidative stress. This is strong family-level inference, not direct KT2440 biochemical proof.	(crack2018redoxsensingiron–sulfurcluster pages 23-25, austin2015aconitasefunctionsas pages 1-2, watanabe2016functionalcharacterizationof pages 1-3)
Regulation / moonlighting	Direct KT2440-specific moonlighting/RNA-binding evidence was not found. In other bacteria, apo-AcnB can become an RNA-binding post-transcriptional regulator after Fe-S cluster loss, so this is a plausible family property but currently unproven for PP_2339 in KT2440. Proteomics in KT2440 shows AcnB abundance remains relatively stable from early to mid exponential growth.	(molina2019pseudomonasputidakt2440 pages 8-9, crack2018redoxsensingiron–sulfurcluster pages 23-25, austin2015aconitasefunctionsas pages 1-2)
Phenotypes / essentiality	No mapped transposon insertions were recovered for PP_2339 in RB-TnSeq studies, interpreted by the authors as suggesting essentiality during library construction. Neighboring MCC genes show strong odd-chain-fatty-acid fitness phenotypes, and authors infer PP_2339 likely provides much of the methylaconitate hydratase activity in this pathway.	(thompson2020fattyacidand pages 5-7, thompson2020functionalanalysisof pages 8-12, thompson2020fattyacidand media b8c40d82)
Localization	No direct localization experiment for PP_2339 was identified in the retrieved evidence. Based on its roles in the TCA/MCC soluble metabolic network and the aconitase enzyme class, AcnB is best inferred to be a cytosolic enzyme rather than membrane or extracellular.	(thompson2020fattyacidand pages 5-7, molina2019pseudomonasputidakt2440 pages 8-9)

Table: This table summarizes the currently supported functional annotation for Pseudomonas putida KT2440 acnB (PP_2339; UniProt Q88KF1). It distinguishes direct KT2440 evidence from broader aconitase-family inference, which is useful where organism-specific biochemical data are limited.

Key visual evidence

Thompson et al. (2020) provides a pathway schematic and gene-fitness heatmap placing AcnB in the methylcitrate cycle used for odd-chain fatty acid catabolism. (thompson2020fattyacidand media b8c40d82, thompson2020fattyacidand media 3b2d456f)

Primary sources (URLs and publication dates)

Thompson MG et al. Applied and Environmental Microbiology (Oct 2020). “Fatty acid and alcohol metabolism in Pseudomonas putida: functional analysis using RB-TnSeq.” https://doi.org/10.1128/aem.01665-20 (thompson2020fattyacidand pages 5-7)
Molina L et al. Environmental Microbiology (Apr 2019). “P. putida KT2440 metabolism undergoes sequential modifications…” https://doi.org/10.1111/1462-2920.14622 (molina2019pseudomonasputidakt2440 pages 8-9)
Moreno R et al. Microbial Biotechnology (Jun 2024). “Inactivation of P. putida KT2440 pyruvate dehydrogenase…” https://doi.org/10.1111/1751-7915.14514 (moreno2024inactivationofpseudomonas pages 1-2)
Crack JC, Le Brun NE. Antioxidants & Redox Signaling (Dec 2018). “Redox-Sensing Iron–Sulfur Cluster Regulators.” https://doi.org/10.1089/ars.2017.7361 (crack2018redoxsensingiron–sulfurcluster pages 23-25)
Austin CM et al. Journal of Bacteriology (Oct 2015). “Aconitase functions as a pleiotropic posttranscriptional regulator in Helicobacter pylori.” https://doi.org/10.1128/JB.00529-15 (austin2015aconitasefunctionsas pages 1-2)
Barrault M et al. Nucleic Acids Research (May 2024). “Staphylococcal aconitase expression during iron deficiency…” https://doi.org/10.1101/2024.05.23.595409 (barrault2024staphylococcalaconitaseexpression pages 1-3)

References

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Artifacts

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Citations

thompson2020fattyacidand pages 5-7
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4Fe-4S
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Functional Annotation Report: AcnB (Aconitate Hydratase B, Q88KF1) in *Pseudomonas putida* KT2440 OpenScientist openscientist-autonomous 9 citations 2 artifacts 2026-07-11T18:29:09.759569

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Functional Annotation Report: AcnB (Aconitate Hydratase B, Q88KF1) in Pseudomonas putida KT2440

Gene: acnB · Locus: PP_2339 · UniProt: Q88KF1
Organism: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950 / KT2440)
EC numbers: 4.2.1.3 (aconitate hydratase) · 4.2.1.99 (2-methylisocitrate dehydratase)
Protein family: Aconitase/IPM isomerase family, AcnB subfamily
Cofactor: Labile catalytic [4Fe-4S] cluster

Summary

AcnB (Q88KF1, PP_2339) is the principal housekeeping aconitase of Pseudomonas putida KT2440 — a cytoplasmic, [4Fe-4S]-cluster-dependent hydro-lyase that catalyzes the reversible, stereospecific isomerization of citrate to isocitrate via the intermediate cis-aconitate (EC 4.2.1.3), the second enzymatic step of the tricarboxylic acid (TCA) cycle. The enzyme uses a catalytic, solvent-exposed, non-cysteine-ligated iron atom of a [4Fe-4S]²⁺ cluster to bind and dehydrate/rehydrate its tricarboxylic-acid substrate. Through the same active site, AcnB also displays a second, physiologically relevant activity: it hydrates 2-methyl-cis-aconitate to 2-methylisocitrate (EC 4.2.1.99), a step of the 2-methylcitrate cycle by which bacteria detoxify and catabolize propionate. This annotation rests on the diagnostic InterPro signatures carried by Q88KF1 (notably the Aconitase 4Fe-4S binding-site signature IPR018136), combined with extensive biochemical and structural characterization of its close orthologs in Escherichia coli and Salmonella enterica.

Confidence in this assignment is exceptionally high because of convergent, orthology-anchored evidence. A global Needleman–Wunsch alignment shows that P. putida AcnB (869 aa) shares 78.3% amino-acid identity with the biochemically and structurally characterized E. coli AcnB (P36683, 865 aa), with near-identical length and shared AcnB-subfamily domain architecture. Critically, 100% of the functionally essential residues are conserved: the three [4Fe-4S]-ligating cysteines (E. coli C710/C769/C772 → P. putida C713/C772/C775) and all ten annotated substrate-binding residues are identical in Q88KF1. This licenses transfer of the detailed E. coli functional characterization to the P. putida enzyme with strong justification.

Beyond catalysis, AcnB is a moonlighting protein that couples central metabolism to iron and redox homeostasis. Its [4Fe-4S] cluster is redox-labile and exists in dynamic equilibrium with the cellular iron pool. Under oxidative stress or iron limitation the cluster is lost, and the resulting apo-protein switches from an enzyme into a post-transcriptional regulator that binds the 3′-untranslated regions (3′UTRs) of acn mRNAs — a bacterial parallel to the eukaryotic aconitase/iron-regulatory-protein-1 (IRP1) "iron-sulfur switch." The enzyme therefore operates entirely within the cytoplasm but performs two distinct jobs depending on the metallation state of its iron-sulfur cluster.

Gene/Protein Identity Verification

The mandatory identity check was completed and passed on all counts:

Verification step	Result
Gene symbol "acnB" matches protein description	✅ UniProt Q88KF1 annotates PP_2339/acnB as "Aconitate hydratase B"
Organism correct	✅ Pseudomonas putida KT2440 (PSEPK)
Protein family/domains align with literature	✅ Aconitase/IPM isomerase family; carries the diagnostic Aconitase 4Fe-4S signature and the AcnB-subfamily domain set
Literature is for the correct gene, not a same-symbol homolog	✅ Orthology confirmed by 78.3% identity to E. coli AcnB and 100% conservation of catalytic residues

The gene symbol acnB is unambiguous in this context: it consistently denotes the "B" isozyme of bacterial aconitase (as opposed to the stress-induced AcnA), and the P. putida protein is a genuine ortholog of the well-studied enterobacterial AcnB. Functional transfer from E. coli/Salmonella is therefore scientifically warranted rather than a same-name confusion.

Note on the evidence base: Direct biochemical/genetic characterization of P. putida KT2440 AcnB specifically is limited in the primary literature. The functional assignment below is anchored on (i) the UniProt/InterPro family annotation for Q88KF1, (ii) strong, well-documented orthology to the extensively characterized E. coli and Salmonella AcnB proteins, and (iii) bioinformatic conservation analyses performed in this investigation. Where a claim rests on the ortholog, that is stated explicitly.

Key Findings

Finding 1 — AcnB is aconitate hydratase B, catalyzing reversible citrate ↔ isocitrate isomerization in the TCA cycle

AcnB is the housekeeping aconitase of P. putida KT2440. Its primary catalytic reaction is the reversible, stereospecific isomerization of citrate to isocitrate, proceeding through the dehydration/rehydration intermediate cis-aconitate. This is the classical aconitase reaction (EC 4.2.1.3) and constitutes the second step of the tricarboxylic acid (Krebs) cycle:

   citrate  ⇌  cis-aconitate + H2O  ⇌  D-threo-isocitrate
     (dehydration)      (rehydration, opposite face)

The reaction is catalyzed at a [4Fe-4S]²⁺ cluster: the substrate coordinates directly to the unique, solvent-exposed iron atom (Fe_a) that is not ligated by protein cysteines, and the cluster mediates removal and re-addition of water across the C–C bond, effecting the net transfer of a hydroxyl group between adjacent carbons.

UniProt Q88KF1 annotates PP_2339/acnB as Aconitate hydratase B, EC 4.2.1.3, and places it in the aconitase/IPM isomerase family bearing the diagnostic Aconitase 4Fe-4S binding-site signature (IPR018136). The functional evidence derives from the biochemically characterized E. coli ortholog. Tang & Guest identified AcnB as "a major but less stable aconitase (AcnB) synthesized during exponential growth" — establishing it as the primary aconitase catalyzing citrate/isocitrate isomerization during active growth PMID: 10589714. Varghese, Tang & Imlay confirmed the catalytic mechanism, noting that "Superoxide damages dehydratases that contain catalytic 4Fe-4S clusters. Aconitases are members of that enzyme family" PMID: 12486059.

This is consistent with the metabolic architecture of P. putida KT2440, which — although it routes glucose catabolism predominantly through the Entner–Doudoroff pathway rather than a complete glycolysis PMID: 26350459 — nonetheless operates a functional TCA cycle for which aconitase activity is indispensable.

Finding 2 — AcnB also acts as 2-methyl-cis-aconitate hydratase (2-methylisocitrate dehydratase, EC 4.2.1.99) in the 2-methylcitrate cycle

Through the same active-site chemistry, AcnB catalyzes the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate (EC 4.2.1.99), a reaction in the 2-methylcitrate cycle — the pathway by which bacteria convert the toxic short-chain fatty acid propionate into pyruvate and succinate for entry into central metabolism:

   2-methylcitrate → 2-methyl-cis-aconitate → 2-methylisocitrate → pyruvate + succinate
(PrpD)              (AcnB / aconitase)      (PrpB, etc.)

UniProt lists this second activity explicitly (EC 4.2.1.99; AltName "2-methylisocitrate dehydratase"). Biochemical reconstitution of the 2-methylcitrate cycle in Salmonella enterica demonstrated that aconitase catalyzes the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate, and that AcnB provides redundant aconitase activity in this pathway: "The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains" PMID: 11294638. Work on Shewanella oneidensis explicitly identified the substrate as belonging to AcnB, describing "2-MCA), a known substrate of the housekeeping aconitase (AcnB" PMID: 29145506. The dual EC assignment therefore reflects genuine catalytic promiscuity for structurally analogous tricarboxylic/methyl-tricarboxylic substrates, arising naturally from the aconitase active site's tolerance of a methyl substituent.

Finding 3 — AcnB is a cytoplasmic, [4Fe-4S]-dependent moonlighting protein: the apo-form binds mRNA and acts as a post-transcriptional regulator

AcnB is a bifunctional (moonlighting) protein. When holo (cluster-loaded) it is a catalytic aconitase; when apo (cluster-lost, under oxidative or iron-limiting stress) it becomes an RNA-binding post-transcriptional regulator. This mirrors the eukaryotic aconitase/IRP1 iron-sulfur switch.

Direct experimental evidence in E. coli shows that "the AcnA and AcnB apo-proteins each interact with the 3′ untranslated regions (3′UTRs) of acnA and acnB mRNA at physiologically significant protein concentrations" PMID: 10589714 — establishing that apo-AcnB binds mRNA and regulates gene expression post-transcriptionally. The trigger for this switch is the lability of the iron-sulfur cluster: "the [4Fe-4S] cluster of AcnB is in dynamic equilibrium with the surrounding iron pool, so that AcnB is rapidly demetallated when intracellular iron pools drop" PMID: 12486059. Cluster maturation and repair are assisted by the dedicated Fe-S carrier protein NfuA, which physically interacts with AcnB: "the ATC* domain interacts with NuoG (a complex I subunit) and aconitase B (AcnB)" PMID: 22966982. The protein carries out both roles in the cytoplasm, consistent with a soluble bacterial central-metabolic enzyme (bacteria have no mitochondrial compartment).

Finding 4 — AcnB belongs to a structurally distinct Gram-negative subfamily with reorganized domain order and an added HEAT-like protein-interaction domain

Structural biology places AcnB in a discrete subfamily. The 2.4 Å crystal structure of E. coli AcnB revealed that, despite a conserved aconitase active site, the enzyme has a reorganized domain order relative to the classic mitochondrial-type aconitase, plus an additional HEAT-like domain unique to the AcnB subfamily. As reported: "the additional domain, characteristic of the AcnB subfamily, is a HEAT-like domain, implying a role in protein protein recognition. This domain packs against the remainder of the protein to form a tunnel leading to the aconitase active site, potentially for substrate channeling" PMID: 11992126.

Q88KF1 carries the full complement of corresponding InterPro domain signatures — IPR001030 (Aconitase/IPM dehydratase large subunit), IPR015928 (Aconitase/3-isopropylmalate dehydratase swivel), IPR015931 (Acnase/IPM dehydratase large subunit alpha/beta/alpha), IPR050926 (Aconitase/IPM isomerase), and IPR018136 (Aconitase 4Fe-4S binding site) — confirming that the P. putida protein shares this AcnB-specific architecture, including the elements that give rise to the HEAT-like domain and the substrate-channeling tunnel.

Finding 5 — Catalysis depends on a redox-sensitive [4Fe-4S]²⁺ cluster that doubles as an iron/redox sensor

The mechanistic heart of AcnB is its redox-sensitive [4Fe-4S]²⁺ cluster. Catalysis proceeds through a solvent-exposed, non-cysteine-ligated iron of the cluster that binds and dehydrates the substrate. Reversible oxidation of the cluster and of nearby cysteine residues switches the enzyme off catalytically and converts the oxidized/apo-form into an iron-regulatory (IRP1-type) protein — a general and well-established property of the aconitase family.

A comprehensive review states plainly that "Catalytic Aco activity is regulated by reversible oxidation of [4Fe-4S]²⁺ cluster and cysteine residues" and that "in the oxidized form it is involved in the control of iron homeostasis as iron regulatory protein 1 (IRP1)" PMID: 24266943. These general principles are directly corroborated by the bacterial AcnB data: superoxide-mediated demetallation and iron-dependent cluster equilibrium are documented specifically for AcnB PMID: 10589714; PMID: 12486059. The cluster's fragility is also demonstrated by toxicological studies showing tellurite-induced, superoxide-dependent disabling of the [4Fe-4S] clusters of E. coli AcnB and fumarase A — over 90% of AcnB activity is lost under oxygen but none under anaerobiosis, with EPR detection of an inactivated [3Fe-4S]⁺ cluster PMID: 19383690 — underscoring how tightly catalysis is coupled to cluster integrity and redox state.

Finding 6 — P. putida AcnB (Q88KF1) is a true ortholog of E. coli AcnB (78% identity), justifying functional transfer

Because most direct biochemistry was performed on the enterobacterial enzyme, establishing rigorous orthology was essential. A global Needleman–Wunsch alignment of full-length P. putida KT2440 AcnB (Q88KF1, 869 aa) against the biochemically and structurally characterized E. coli AcnB (P36683, ACNB_ECOLI, 865 aa) yields 690/881 identical positions = 78.3% identity over the alignment (79.8% over the shorter sequence), with near-identical length. Both proteins are annotated "Aconitate hydratase B" and share the AcnB-subfamily domain architecture. An identity of ~78% over the entire length of an 869-residue enzyme far exceeds the threshold at which enzyme function and mechanism are conserved, making the transfer of E. coli functional characterization to the P. putida enzyme highly reliable.

Property	P. putida AcnB (Q88KF1)	E. coli AcnB (P36683)
Length	869 aa	865 aa
Annotation	Aconitate hydratase B	Aconitate hydratase B
Global identity	—	78.3% (690/881 aligned positions)
Subfamily	AcnB (HEAT-like domain)	AcnB (HEAT-like domain)
EC numbers	4.2.1.3 / 4.2.1.99	4.2.1.3 / 4.2.1.99

Finding 7 — All catalytic residues are 100% conserved in Q88KF1

The strongest single line of evidence is residue-level conservation. Mapping the UniProt-annotated functional residues of E. coli AcnB (P36683) onto Q88KF1 via the global alignment shows perfect conservation of every catalytically essential position:

Functional role	E. coli AcnB residue(s)	P. putida AcnB residue(s)	Conserved?
[4Fe-4S] cluster-ligating cysteine 1	C710	C713	✅
[4Fe-4S] cluster-ligating cysteine 2	C769	C772	✅
[4Fe-4S] cluster-ligating cysteine 3	C772	C775	✅
Substrate binding	R191	R191	✅
Substrate binding	S244, S245, R246	S244, S245, R246	✅
Substrate binding	Q414, D415, T416	Q417, D418, T419	✅
Substrate binding	S498	S501	✅
Substrate binding	R791, R796	R794, R799	✅

The three cysteines that ligate the [4Fe-4S] cluster and all ten annotated substrate-binding residues are identical in the P. putida enzyme. This level of conservation — with only a small, consistent numbering offset from a short insertion — is dispositive: the P. putida enzyme possesses an intact, canonical aconitase active site and iron-sulfur cluster binding site, confirming it is a fully functional aconitase B.

Mechanistic Model / Interpretation

The findings integrate into a single coherent model in which the metallation and redox state of one [4Fe-4S] cluster toggles AcnB between two mutually exclusive functions:

          ┌──────────────────────────────────────────────┐
          │            AcnB (Q88KF1, PP_2339)             │
          │      cytoplasmic, 869 aa, AcnB subfamily      │
          └──────────────────────────────────────────────┘
                          │
 ┌────────────────────────────────┴─────────────────────────────────┐
 │                                                                    │
   HOLO-form  [4Fe-4S]²⁺ intact                          APO-form  (cluster lost)
   ── CATALYTIC ENZYME ──                                ── RNA-BINDING REGULATOR ──
 │                                                                    │
   ┌─────┴──────────────────────────┐                        ┌───────────────┴───────────────┐
   │ EC 4.2.1.3 (TCA cycle):        │                        │ Binds 3'UTR of acnA/acnB mRNA  │
   │   citrate ⇌ cis-aconitate      │                        │ Post-transcriptional control    │
   │           ⇌ isocitrate         │                        │ IRP1-like "iron-sulfur switch"  │
   │                                │                        └────────────────────────────────┘
   │ EC 4.2.1.99 (2-MC cycle):      │                                       ▲
   │   2-methyl-cis-aconitate       │        Trigger: oxidative stress (O2•⁻),
   │      → 2-methylisocitrate      │        iron limitation → cluster demetallation
   └────────────────────────────────┘        (assisted repair by NfuA carrier)

Direction of the switch. Under nutrient-replete, low-stress conditions, AcnB is loaded with its [4Fe-4S]²⁺ cluster and runs as the dominant exponential-phase aconitase, driving carbon through the TCA cycle and — when propionate is present — through the 2-methylcitrate cycle. The active-site iron atom (Fe_a) that is not held by a protein cysteine is the catalytic centre: it coordinates the substrate's hydroxyl/carboxylate and activates water for the dehydration–rehydration that interconverts citrate and isocitrate. Because the same geometry accommodates a methyl-substituted substrate, the enzyme also processes 2-methyl-cis-aconitate.

The sensing logic. The cluster's fourth iron is deliberately labile, placing the whole cluster in dynamic equilibrium with the cytoplasmic "chelatable" iron pool and rendering it acutely sensitive to superoxide. When iron drops or oxidative stress rises, the cluster disassembles, catalysis stops, and the protein's fold shifts to expose an RNA-binding surface. Apo-AcnB then binds the 3′UTRs of acn transcripts, coupling the cell's central-metabolic flux capacity to its iron and redox status. NfuA acts as the repair valve, delivering/rebuilding Fe-S clusters to restore the catalytic state when conditions improve. This is a bacterial homolog of the eukaryotic cytosolic aconitase/IRP1 system, in which the identical switch controls iron homeostasis PMID: 24266943.

Why this matters for P. putida. P. putida KT2440 is a metabolically robust soil bacterium that thrives under oxidative and nutritional stress, partly by maintaining a catabolic overproduction of NADPH reducing power through its EDEMP cycle PMID: 26350459. An aconitase whose activity is directly gated by redox/iron status fits neatly into this stress-resilient physiology: AcnB is simultaneously a workhorse of oxidative central metabolism and a sentinel that throttles that metabolism — and communicates the alarm at the mRNA level — when the cell's redox buffering is challenged.

Evidence Base

PMID	Title (abbrev.)	How it supports the annotation
10589714	Direct evidence for mRNA binding and post-transcriptional regulation by E. coli aconitases	Identifies AcnB as the major exponential-phase aconitase (F1); demonstrates apo-AcnB binds acn mRNA 3′UTRs (F3)
12486059	Contrasting sensitivities of E. coli aconitases A and B to oxidation and iron depletion	Establishes catalytic [4Fe-4S]²⁺ mechanism (F1); shows cluster in dynamic equilibrium with iron pool, driving the switch (F3, F5)
11294638	In vitro conversion of propionate to pyruvate by Salmonella enzymes	Shows AcnB provides redundant aconitase activity hydrating 2-methyl-cis-aconitate → 2-methylisocitrate (F2)
29145506	PrpF of Shewanella catalyzes isomerization of 2-methyl-cis-aconitate	Explicitly names 2-methyl-cis-aconitate as a substrate of housekeeping aconitase AcnB (F2)
11992126	E. coli aconitase B structure reveals a HEAT-like domain	Defines the AcnB subfamily's reorganized domains and HEAT-like protein-interaction domain forming a substrate-channeling tunnel (F4)
22966982	Molecular organization, function, role and evolution of NfuA, an atypical Fe-S carrier	Shows the Fe-S carrier NfuA physically interacts with AcnB to mature/repair its cluster (F3)
24266943	Aconitase post-translational modification linking Krebs cycle, iron homeostasis, redox signaling	Establishes redox-sensitive [4Fe-4S]²⁺ cluster as catalytic basis and IRP1 regulatory switch (F5)
19383690	Tellurite-mediated disabling of [4Fe-4S] clusters of E. coli dehydratases	Demonstrates superoxide-dependent, O₂-dependent disabling of AcnB's [4Fe-4S] cluster (supports F5)
26350459	P. putida KT2440 metabolizes glucose through the EDEMP cycle	Provides the P. putida central-metabolic context in which AcnB's TCA-cycle role operates
20053667	Molecular control of the cytosolic aconitase/IRP1 switch by extramitochondrial frataxin	Frames the moonlighting aconitase/IRP1 iron-sulfur switch generic to the family

Bioinformatic evidence generated in this investigation (Findings 6 and 7): global pairwise alignment of Q88KF1 vs. P36683 (78.3% identity, 690/881 positions) and residue-level mapping showing 100% conservation of the three Fe-S-ligating cysteines and all ten substrate-binding residues. These computations anchor the orthology and legitimize functional transfer from the enterobacterial literature to the P. putida protein.

Convergence. Four independent evidence streams point to the same conclusion: (1) InterPro/UniProt domain signatures; (2) direct biochemistry and structure of the E. coli/Salmonella AcnB orthologs; (3) 78% full-length sequence identity establishing orthology; and (4) 100% conservation of catalytic and cluster-ligating residues. No conflicting evidence was encountered.

Supported vs. Refuted Hypotheses

Hypothesis	Status	Basis
AcnB is the TCA-cycle aconitase (citrate⇌isocitrate)	Supported	UniProt EC 4.2.1.3; ortholog housekeeping aconitase (PMID 10589714)
AcnB also acts as 2-methylisocitrate dehydratase (EC 4.2.1.99)	Supported	UniProt AltName; PMID 11294638, 29145506
AcnB uses a catalytic [4Fe-4S] cluster	Supported	IPR018136; PMID 12486059, 19383690
AcnB is cytoplasmic and bifunctional (moonlighting mRNA-binding)	Supported	PMID 10589714, 12486059, 22966982
Q88KF1 is a true AcnB ortholog (not a paralog)	Supported	78.3% global identity to E. coli AcnB (this work)
Q88KF1 has an intact aconitase active site + Fe-S site	Supported	3/3 Fe-S Cys + 10/10 substrate residues 100% conserved (this work)
The gene symbol is ambiguous / mis-assigned	Refuted	Symbol, family, domains, EC, and 78% ortholog identity all align

Limitations and Knowledge Gaps

No direct P. putida experimental data. The functional assignment rests entirely on high-confidence homology transfer from E. coli and Salmonella orthologs plus bioinformatic conservation analysis. No enzymatic assay, crystal structure, knockout phenotype, or RNA-binding assay has been performed on the P. putida AcnB protein itself. While the ~78% identity and 100% active-site conservation make functional divergence extremely unlikely, this remains inference rather than direct measurement in the target organism.
Kinetic parameters unknown for the target. Substrate affinities (K_m), turnover numbers (k_cat), and the relative catalytic efficiency for citrate/isocitrate versus 2-methyl-cis-aconitate have not been measured for Q88KF1. The quantitative balance between the EC 4.2.1.3 and EC 4.2.1.99 activities in P. putida is therefore unquantified.
Regulatory role not confirmed in P. putida. The moonlighting mRNA-binding function is documented in E. coli. Whether P. putida AcnB binds specific transcripts, and which ones, has not been established; the 3′UTR targets in P. putida may differ from those in E. coli.
2-methylcitrate cycle context in P. putida. The precise composition and gene organization of the P. putida propionate-catabolism (2-methylcitrate) pathway, and AcnB's exact contribution relative to any dedicated PrpD/AcnD-type enzymes, has not been mapped in this investigation.
Subcellular localization inferred, not measured. Cytoplasmic localization is inferred from the soluble nature of bacterial aconitases and the absence of signal/localization sequences; it has not been experimentally verified for the P. putida protein (though it is essentially certain).
Structure is homology-based. No experimental structure of Q88KF1 exists; structural claims rest on the E. coli AcnB crystal structure and conserved domain signatures.

Proposed Follow-up Experiments / Actions

Recombinant enzyme kinetics. Express and purify Q88KF1, reconstitute the [4Fe-4S] cluster anaerobically, and measure aconitase activity (citrate ⇌ isocitrate) and 2-methylisocitrate dehydratase activity to determine K_m/k_cat for each substrate and quantify the relative catalytic efficiencies.
Gene knockout phenotyping. Construct a P. putida KT2440 ΔacnB (ΔPP_2339) mutant and assess growth on TCA-cycle-dependent carbon sources and on propionate. Test for synthetic phenotypes with any acnA-type paralog to probe functional redundancy, mirroring the Salmonella observations.
Cluster lability and redox response. Measure loss of AcnB activity in cells and in vitro under superoxide stress and iron limitation to confirm the redox/iron-gated switch operates in P. putida as in E. coli.
RNA-binding assay. Test whether apo-Q88KF1 binds candidate 3′UTRs (e.g., its own transcript) by electrophoretic mobility shift or RNA immunoprecipitation, to determine whether the IRP1-like moonlighting function is conserved in P. putida.
Structural determination. Solve a crystal or cryo-EM structure of Q88KF1 (or generate a high-quality AlphaFold model) to confirm the AcnB-subfamily fold, the HEAT-like domain, and the substrate-channeling tunnel, and to visualize the intact active site.
Interactomics. Test the predicted physical interaction between P. putida AcnB and its NfuA-type Fe-S carrier to confirm the cluster maturation/repair pathway.
Propionate-pathway mapping. Delineate the P. putida 2-methylcitrate operon and AcnB's role within it, using transcriptomics/proteomics under propionate growth.

Conclusion

AcnB (Q88KF1, PP_2339) is confidently annotated as aconitate hydratase B, the principal cytoplasmic housekeeping aconitase of Pseudomonas putida KT2440. It catalyzes the reversible, stereospecific isomerization of citrate to isocitrate via cis-aconitate (EC 4.2.1.3) using a catalytic, redox-labile [4Fe-4S]²⁺ cluster, and through the same active site hydrates 2-methyl-cis-aconitate to 2-methylisocitrate (EC 4.2.1.99) in the 2-methylcitrate/propionate-catabolism cycle. It acts in the cytoplasm and is a moonlighting protein whose apo-form, generated under oxidative or iron-limiting stress, binds mRNA and acts as an IRP1-like post-transcriptional regulator. This assignment is supported by convergent evidence: diagnostic aconitase/4Fe-4S InterPro signatures, extensive experimental characterization of the E. coli/Salmonella AcnB orthologs, 78.3% full-length identity to E. coli AcnB, and 100% conservation of the three Fe-S-ligating cysteines and all ten substrate-binding residues in Q88KF1.

References

Tang Y, Guest JR (1999). Direct evidence for mRNA binding and post-transcriptional regulation by E. coli aconitases. PMID 10589714
Varghese S, Tang Y, Imlay JA (2003). Contrasting sensitivities of E. coli aconitases A and B to oxidation and iron depletion. PMID 12486059
Williams CH et al. (2002). E. coli aconitase B structure reveals a HEAT-like domain with implications for protein-protein recognition. PMID 11992126
Horswill AR, Escalante-Semerena JC (2001). In vitro conversion of propionate to pyruvate by S. enterica enzymes (PrpD and aconitase). PMID 11294638
Rocco CJ et al. (2017). PrpF of S. oneidensis MR-1 catalyzes isomerization of 2-methyl-cis-aconitate in the AcnD-dependent 2-methylcitric acid cycle. PMID 29145506
Py B et al. (2012). Molecular organization, biochemical function, cellular role and evolution of NfuA, an atypical Fe-S carrier. PMID 22966982
Calderón IL et al. (2009). Tellurite-mediated disabling of [4Fe-4S] clusters of E. coli dehydratases. PMID 19383690
Nikel PI et al. (2015). P. putida KT2440 metabolizes glucose through the EDEMP cycle. PMID 26350459
Lushchak OV et al. (2014). Aconitase post-translational modification as a key in linkage between Krebs cycle, iron homeostasis, redox signaling and ROS metabolism. PMID 24266943
Condò I et al. (2010). Molecular control of the cytosolic aconitase/IRP1 switch by extramitochondrial frataxin. PMID 20053667

Artifacts

Citations

PMID:10589714
PMID:12486059
PMID:26350459
PMID:11294638
PMID:29145506
PMID:22966982
PMID:11992126
PMID:24266943
PMID:19383690

acnB

Gene Description

Existing Annotations Review

Core Functions

Aconitate hydratase (aconitase) activity catalyzing reversible citrate-cis-aconitate-isocitrate isomerization in the TCA cycle, dependent on a catalytic [4Fe-4S] cluster.

Aconitase-family hydration of 2-methyl-cis-aconitate to 2-methylisocitrate in the methylcitrate cycle for propionate catabolism.

References

Deep Research

Falcon

Research Report: Functional Annotation of acnB (UniProt Q88KF1; locus PP_2339) in Pseudomonas putida KT2440

0. Target verification (critical disambiguation)

1. Key concepts and current definitions

1.1 Aconitase / aconitate hydratase (EC 4.2.1.3)

1.2 Methylcitrate cycle (MCC) and “bifunctional” AcnB

1.3 Apo-aconitase “moonlighting” as an RNA-binding protein (general bacterial concept)

2. Functional role of P. putida KT2440 AcnB (PP_2339; Q88KF1)

2.1 Primary biochemical function: central-carbon dehydratase/isomerase chemistry

2.2 Pathway context: methylcitrate cycle is required for odd-chain fatty acid utilization

2.3 Cellular localization

3. Phenotypes, essentiality, and regulation in P. putida KT2440

3.1 Essentiality / genetic constraint

3.2 Expression/proteomic behavior in growth phases

3.3 Oxidative stress / iron limitation considerations (evidence boundaries)

4. Recent developments (prioritizing 2023–2024)

4.1 2023: genome-scale functional genetics resources for KT2440

4.2 2024: applied central-metabolism rewiring for aromatic compound degradation

4.3 2024: authoritative demonstration of iron-stress aconitase moonlighting (cross-species mechanistic advance)

5. Current applications and real-world implementations

5.1 Bioremediation/biotransformation requiring robust central metabolism

5.2 Odd-chain fatty acid utilization and propionyl-CoA detoxification

6. Relevant statistics and data (from available sources)

7. Expert analysis and synthesis

7.1 Most defensible functional annotation for Q88KF1 in KT2440

7.2 Why “bifunctional” matters in P. putida

7.3 Stress biology: what can be inferred vs what is proven

Embedded summary table

Key visual evidence

Primary sources (URLs and publication dates)

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Citations

OpenScientist

Functional Annotation Report: AcnB (Aconitate Hydratase B, Q88KF1) in Pseudomonas putida KT2440

Summary

Gene/Protein Identity Verification

Key Findings

Finding 1 — AcnB is aconitate hydratase B, catalyzing reversible citrate ↔ isocitrate isomerization in the TCA cycle

Finding 2 — AcnB also acts as 2-methyl-cis-aconitate hydratase (2-methylisocitrate dehydratase, EC 4.2.1.99) in the 2-methylcitrate cycle

Finding 3 — AcnB is a cytoplasmic, [4Fe-4S]-dependent moonlighting protein: the apo-form binds mRNA and acts as a post-transcriptional regulator

Finding 4 — AcnB belongs to a structurally distinct Gram-negative subfamily with reorganized domain order and an added HEAT-like protein-interaction domain

Finding 5 — Catalysis depends on a redox-sensitive [4Fe-4S]²⁺ cluster that doubles as an iron/redox sensor

Finding 6 — P. putida AcnB (Q88KF1) is a true ortholog of E. coli AcnB (78% identity), justifying functional transfer

Finding 7 — All catalytic residues are 100% conserved in Q88KF1

Mechanistic Model / Interpretation

Evidence Base

Supported vs. Refuted Hypotheses

Limitations and Knowledge Gaps

Proposed Follow-up Experiments / Actions

Conclusion

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