icd

UniProt ID: Q88FS2
Organism: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950 / KT2440)
Review Status: DRAFT
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Gene Description

NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) of Pseudomonas putida KT2440 (locus PP_4011). It catalyzes the divalent-metal-dependent (Mg2+/Mn2+) oxidative decarboxylation of D-threo-isocitrate to 2-oxoglutarate and CO2 with the concomitant reduction of NADP+ to NADPH. The enzyme is a soluble cytoplasmic homodimer belonging to the isocitrate/isopropylmalate dehydrogenase family. It acts at the isocitrate node of the tricarboxylic acid cycle, where it both supplies 2-oxoglutarate for amino-acid biosynthesis and is a major source of anabolic reducing power (NADPH) for central metabolism and redox balance. In KT2440, biochemical assays of cell-free extracts show a strong (~89%) preference for NADP+ over NAD+. Activity at this branch point is subject to post-translational regulation (phosphorylation of a conserved serine), partitioning carbon between the TCA cycle and the glyoxylate shunt.

Existing Annotations Review

GO Term Evidence Action Reason
GO:0000287 magnesium ion binding
IEA
GO_REF:0000002 		ACCEPT
Summary: NADP-IDH requires a divalent metal ion (Mg2+ or Mn2+) per subunit for catalysis, and UniProt annotates a Mg2+-binding residue (position 309). Magnesium ion binding is well supported for this family.
Reason: Consistent with the IDH/IMDH family requirement for a divalent metal cofactor; UniProt records both Mg2+ and Mn2+ cofactors and a Mg2+-binding site.
GO:0004450 isocitrate dehydrogenase (NADP+) activity
IEA
GO_REF:0000120 		ACCEPT
Summary: This is the core molecular function. The enzyme catalyzes oxidative decarboxylation of D-threo-isocitrate to 2-oxoglutarate + CO2 with reduction of NADP+ (EC 1.1.1.42, RHEA:19629). KT2440-specific biochemistry confirms strong NADP+ preference (~89% NADP+ vs ~11% NAD+ in cell-free extracts).
Reason: Directly matches UniProt catalytic activity and is corroborated by organism-specific biochemical measurements of NADP cofactor preference (Nikel et al. 2015, PMID:26350459).
GO:0006099 tricarboxylic acid cycle
IEA
GO_REF:0000120 		ACCEPT
Summary: Isocitrate dehydrogenase catalyzes the isocitrate to 2-oxoglutarate step of the TCA cycle. This is the appropriate biological process for the core function.
Reason: Standard, well-supported placement of IDH within the TCA cycle; consistent with central carbon metabolism studies in KT2440.
GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor
IEA
GO_REF:0000002 		MARK AS OVER ANNOTATED
Summary: This is a generic parent term of the specific and more informative GO:0004450 (isocitrate dehydrogenase (NADP+) activity), which is already annotated. It is not incorrect but adds no information beyond the specific child.
Reason: Redundant grandparent of the specific MF term GO:0004450 that is already present; provides no additional specificity.
GO:0051287 NAD binding
IEA
GO_REF:0000002 		REMOVE
Summary: This enzyme is NADP+-specific, not NAD+-binding. UniProt annotates multiple NADP(+)-binding residues (positions 106, 341-347, 354, 393, 397) and no NAD-binding site, and KT2440 biochemistry shows ~89% NADP+ preference. The InterPro2GO mapping to NAD binding is a mis-propagation for this NADP-specific family member; the correct cofactor binding is NADP, not NAD.
Reason: Contradicted by cofactor specificity. The enzyme binds NADP+, not NAD+; this is an over-propagated InterPro IEA inference. NADP binding (GO:0050661) would be the correct term.
Proposed replacements: NADP binding

Core Functions

NADP-dependent isocitrate dehydrogenase that catalyzes the divalent-metal-dependent oxidative decarboxylation of D-threo-isocitrate to 2-oxoglutarate and CO2, reducing NADP+ to NADPH, within the TCA cycle.

Molecular Function:
isocitrate dehydrogenase (NADP+) activity
Directly Involved In:
tricarboxylic acid cycle
Supporting Evidence:
  • PMID:26350459
    KT2440 cell-free extract assays show isocitrate dehydrogenase with strong (~89%) preference for NADP+ over NAD+, identifying it as an NADPH-forming dehydrogenase in central carbon metabolism.

References

GO_REF:0000002
Gene Ontology annotation through association of InterPro records with GO terms
GO_REF:0000120
Combined Automated Annotation using Multiple IEA Methods
PMID:26350459
Pseudomonas putida KT2440 strain metabolizes glucose through a cycle formed by enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways
  • In KT2440 cell-free extracts, isocitrate dehydrogenase (Icd/PP_4011) shows ~89% activity with NADP+ versus ~11% with NAD+, confirming NADP+ specificity and a role in NADPH supply.
PMID:30936206
Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida
  • Phosphorylation of the ICD-associated protein PP_4011 changes with carbon source, consistent with post-translational tuning of flux at the TCA/glyoxylate branch point.

Deep Research

Falcon

(icd-deep-research-falcon.md)
Research Report: Functional Annotation of **icd** (UniProt **Q88FS2**, locus **PP_4011**) in *Pseudomonas putida* KT2440 Falcon Edison Scientific Literature 22 citations 2 artifacts 2026-06-11T21:22:31.319845
Edison artifact artifact-00 (text/markdown)
## Context ID: pqac-00000004 The table reporting cofactor specificity for dehydrogenases in Pseudomonas putida KT2440 is Table 3 on page 21. For isocitrate dehy (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 icd (UniProt Q88FS2, locus PP_4011) in Pseudomonas putida KT2440

0. Scope, identity verification, and ambiguity control

This report concerns icd (PP_4011) from Pseudomonas putida strain KT2440 (ATCC 47054/DSM 6125/NCIMB 11950), UniProt accession Q88FS2, annotated as isocitrate dehydrogenase [NADP] (EC 1.1.1.42).

The gene/protein identity is supported by P. putida KT2440-specific literature that explicitly labels icd/PP_4011 as isocitrate dehydrogenase and measures its NADP vs NAD cofactor preference in KT2440 extracts (nikel2015pseudomonasputidakt2440 pages 21-25, nikel2015pseudomonasputidakt2440 media 99f494ff). A separate KT2440 multi-omics study also refers to PP_4011 as an “ICD-associated protein” in phosphorylation analyses (kukurugya2019multiomicsanalysisunravels pages 10-11). These match the UniProt-provided description (NADP-IDH; EC 1.1.1.42; central carbon metabolism enzyme), so the report does not conflate this icd with similarly named genes from other organisms.

1. Key concepts and current understanding

1.1 Definition and primary biochemical function

Isocitrate dehydrogenase (IDH/ICD; NADP-dependent; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate (α-ketoglutarate), producing CO2 and reduced pyridine nucleotide. In the NADP-dependent form, the physiological product is NADPH, linking the TCA cycle to anabolic reducing power.

In KT2440, ICD is treated as a major intracellular dehydrogenase contributing to redox metabolism; a systematic enzyme survey concluded ICD has >80% specificity for NADP+ over NAD+ (nikel2015pseudomonasputidakt2440 pages 7-8).

1.2 Cofactor specificity determinants (conceptual background)

Bacterial “type I” homodimeric IDHs vary in cofactor usage (NADP-specific, NAD-specific, or dual-specific). Sequence/structure comparisons indicate that specific residues in the coenzyme-binding pocket determine whether NADP’s 2′-phosphate is stabilized (favoring NADP) or disfavored (favoring NAD). Romkina & Kiriukhin (2017) summarize motifs associated with NADP specificity (e.g., Lys/Tyr/Val positions) and how substitutions (e.g., Lys→Asp) can shift preference toward NAD (romkina2017biochemicalandmolecular pages 7-9).

1.3 Canonical regulation at the TCA–glyoxylate branchpoint

In several bacteria (classically E. coli), ICD activity can be reversibly controlled by AceK (isocitrate dehydrogenase kinase/phosphatase), which phosphorylates a conserved serine on ICD, decreasing activity and redirecting carbon from the TCA cycle into the glyoxylate shunt. This mechanism is summarized in Romkina & Kiriukhin (2017) as an established post-translational switch controlling flux partitioning (romkina2017biochemicalandmolecular pages 7-9).

2. Gene/protein function in P. putida KT2440

2.1 Reaction and substrate specificity

The substrate is isocitrate; the product is 2-oxoglutarate (α-ketoglutarate), with concurrent CO2 release and reduction of NADP+ to NADPH. While the retrieved KT2440-focused texts emphasize cofactor preference rather than Km/kcat values, they repeatedly interpret ICD as an NADPH-forming dehydrogenase within central metabolism and redox balance (nikel2015pseudomonasputidakt2440 pages 7-8, kukurugya2019multiomicsanalysisunravels pages 10-11).

2.2 Cofactor specificity: quantitative measurements in KT2440

In cell-free extracts of exponentially growing KT2440 on glucose, Icd (PP_4011) shows strong NADP preference. Under saturating conditions, relative activity was 88.5 ± 6.6% with NADP+ vs 11.5 ± 1.9% with NAD+; under non-saturating “quasi in vivo” conditions, 88.9 ± 2.3% (NADP+) vs 11.1 ± 0.9% (NAD+) (nikel2015pseudomonasputidakt2440 pages 21-25, nikel2015pseudomonasputidakt2440 media 99f494ff). This supports annotation as NADP-dependent and indicates its principal physiological role is NADPH generation.

2.3 Cellular localization

ICD (Icd/PP_4011) is treated as a soluble intracellular enzyme in central metabolism: it is assayed from cell-free extracts and discussed as part of cytosolic flux through the TCA/glyoxylate node rather than periplasmic oxidation (nikel2015pseudomonasputidakt2440 pages 21-25, kukurugya2019multiomicsanalysisunravels pages 10-11). Thus, the most evidence-supported localization is cytoplasmic.

2.4 Pathway context: TCA cycle, glyoxylate shunt, and redox balance

A KT2440 ^13C/enzymology study describes a cyclic architecture integrating ED/EMP/PPP (“EDEMP cycle”) for glucose catabolism and provides cofactor-specificity measurements for multiple dehydrogenases including ICD (nikel2015pseudomonasputidakt2440 pages 7-8, nikel2015pseudomonasputidakt2440 pages 21-25). In this framework, ICD is one of the intracellular nodes contributing to NADPH supply, complementing NADPH generation in oxidative PPP and other dehydrogenase steps (nikel2015pseudomonasputidakt2440 pages 7-8).

A multi-omics study on glucose plus benzoate co-utilization highlighted the ICD node as a regulatory point in mixed-substrate metabolism, noting changes in phosphorylation of PP_4011 and interpreting these changes as part of maintaining flux directionality/magnitude and redox demands around the TCA–glyoxylate branch (kukurugya2019multiomicsanalysisunravels pages 10-11).

3. Regulation and control of ICD activity in KT2440

3.1 Phosphorylation-based regulation in mixed-substrate metabolism (primary evidence)

Kukurugya et al. (2019) report a decrease in phosphorylation of an “ICD-associated protein (PP_4011)” in KT2440 during growth on a glucose:benzoate mixture vs glucose alone (kukurugya2019multiomicsanalysisunravels pages 10-11). They suggest this phosphorylation change may help counteract “overwhelming metabolite-level inhibition” of ICD activity expected from glyoxylate shunt metabolites and pyruvate accumulation (kukurugya2019multiomicsanalysisunravels pages 10-11). This provides direct organism-specific evidence that phosphorylation state at/around PP_4011 changes with carbon source context.

3.2 AceK-mediated routing hypothesis in 2024 electrogenic/anoxic systems biology

A 2024 systems metabolic engineering analysis of electrogenic/anoxic KT2440 links elevated acetyl-CoA to AceK activity, describing AceK as phosphorylating and partially inactivating ICD, thereby redirecting carbon flux toward the glyoxylate shunt (weimer2024systemsmetabolicengineeringa pages 69-74). This is presented as a mechanistic interpretation of multi-omics shifts under bio-electrochemical conditions and places icd/ICD within modern regulatory models for non-canonical growth/production states (weimer2024systemsmetabolicengineeringa pages 69-74).

4. Recent developments (prioritizing 2023–2024)

4.1 2024: Adaptation to non-native xylose and redox mapping that includes ICD

Dvořák et al. (Nature Communications, March 2024) investigated engineered/ALE adaptation of P. putida to D-xylose and mapped redox-producing steps. Their analysis explicitly treats overproduction of reducing cofactors (NAD(P)H) as a driver of pathway routing; ICD is included in the central carbon metabolism mapping and annotated as producing “CO2 NADPH” at the isocitrate node (dvorak2024syntheticallyprimedadaptationof pages 3-4). While the excerpted text does not give an explicit icd fold-change, it situates ICD within current (2024) systems-level understanding of how redox supply constrains pathway use in engineered KT2440 backgrounds (dvorak2024syntheticallyprimedadaptationof pages 3-4).

4.2 2024: Electrogenic/anoxic bioprocessing and ICD-linked flux partitioning

Weimer et al. (Microbial Cell Factories, September 2024) reported systems-level characterization of an electrogenic anoxic phenotype of KT2440 and engineered improved 2-ketogluconate (2KG) production. They report a best-case 2KG yield of 0.96 mol/mol glucose in an engineered mutant background under these conditions (weimer2024systemsmetabolicengineeringa pages 17-21). A related 2024 systems metabolic engineering narrative links this regime to glyoxylate shunt routing via AceK–ICD modulation (weimer2024systemsmetabolicengineeringa pages 69-74).

5. Current applications and real-world implementations

5.1 Metabolic engineering target for biopolymer (PHA) production

Zhou et al. (Communications Biology, March 2020) used icd (PP_4011) as a genome-editing demonstration target in a CRISPR/Cas9n-λ-Red method for KT2440. They note prior proposals that icd inactivation could increase acetyl-CoA flux into fatty-acid biosynthesis (a rationale relevant to PHA synthesis) (zhou2020developmentofa pages 7-9). In their specific engineered background, however, they report that icd deletion did not contribute to mcl-PHA synthesis (zhou2020developmentofa pages 7-9). Importantly for functional annotation, they achieved scarless deletion and sequencing confirmation for the icd locus in multiple isolates, indicating genetic tractability and viability under their lab conditions (zhou2020developmentofa pages 4-6).

5.2 Systems-level strain design and redox-intensive bioprocess states

Recent KT2440 engineering efforts often aim to exploit or reshape NADPH supply. ICD is repeatedly positioned as a key NADPH-forming step connected to the TCA/glyoxylate switch, relevant to redox balance during mixed-substrate utilization and specialized production regimes (kukurugya2019multiomicsanalysisunravels pages 10-11, weimer2024systemsmetabolicengineeringa pages 69-74).

6. Expert interpretation and analysis (grounded in cited sources)

  1. Primary functional role: The strongest KT2440-specific evidence supports ICD (PP_4011) as an NADP-preferring intracellular IDH whose physiological output is NADPH (nikel2015pseudomonasputidakt2440 pages 21-25, nikel2015pseudomonasputidakt2440 media 99f494ff). This aligns with its expected central metabolic role at the isocitrate→2-oxoglutarate step.
  2. Regulatory importance: Multi-omics evidence indicates that phosphorylation state at/around PP_4011 changes with carbon-source context, consistent with the broader concept that phosphorylation-based switches at ICD tune flux at the TCA/glyoxylate branchpoint (kukurugya2019multiomicsanalysisunravels pages 10-11, romkina2017biochemicalandmolecular pages 7-9).
  3. Engineering relevance: Although icd deletion was feasible and historically proposed as a lever to redirect acetyl-CoA to lipid/PHA synthesis, at least one KT2440 engineering study found no benefit for mcl-PHA under its conditions, illustrating that icd’s effect is context-dependent and likely buffered by network-level redox/flux control (zhou2020developmentofa pages 7-9).

7. Key statistics and data points (recent and foundational)

  • Cofactor specificity (KT2440 Icd/PP_4011): ~89% NADP vs ~11% NAD activity in extracts (both saturating and quasi in vivo assays) (nikel2015pseudomonasputidakt2440 pages 21-25, nikel2015pseudomonasputidakt2440 media 99f494ff).
  • Mixed-substrate (glucose:benzoate) systems data around ICD node: >10-fold increase in acetyl-CoA reported in the same study that observed reduced phosphorylation of PP_4011; and a 37% increase in NAD(P)H yield in glucose-only versus glucose:benzoate condition (kukurugya2019multiomicsanalysisunravels pages 10-11).
  • 2024 electrogenic/anoxic bioprocess metric: 2KG yield reported up to 0.96 mol/mol in engineered KT2440 under bio-electrochemical anoxic conditions (weimer2024systemsmetabolicengineeringa pages 17-21).
  • CRISPR/Cas9n engineering statistic: For the icd locus, authors report precise scarless deletion confirmed by sequencing of 15/15 tested mutants (zhou2020developmentofa pages 4-6).

8. Evidence summary table

The following table consolidates key annotation-relevant facts and quantitative measurements.

Feature Summary for Pseudomonas putida KT2440 icd (PP_4011; UniProt Q88FS2) Evidence
Gene/protein identity icd / PP_4011 encodes isocitrate dehydrogenase [NADP], a bacterial type I IDH/ICD in central carbon metabolism; the literature explicitly maps icd/PP_4011 to isocitrate dehydrogenase in P. putida KT2440. (nikel2015pseudomonasputidakt2440 pages 21-25)
Enzyme name and EC Isocitrate dehydrogenase (NADP-dependent), EC 1.1.1.42. This enzyme belongs to the TCA-cycle oxidative decarboxylation step that converts isocitrate to 2-oxoglutarate while reducing NADP+. (nikel2015pseudomonasputidakt2440 pages 7-8, nikel2015pseudomonasputidakt2440 pages 21-25)
Reaction catalyzed Catalyzes the oxidative decarboxylation of isocitrate + NADP+ → 2-oxoglutarate + CO2 + NADPH; in KT2440 it is discussed as a major NADPH-generating dehydrogenase connected to the TCA cycle and redox balance. (nikel2015pseudomonasputidakt2440 pages 7-8, kukurugya2019multiomicsanalysisunravels pages 10-11)
Cofactor specificity Strongly NADP-preferring rather than NAD-specific. A broader biochemical survey of KT2440 dehydrogenases states that isocitrate dehydrogenase shows >80% specificity for NADP+ over NAD+. (nikel2015pseudomonasputidakt2440 pages 7-8)
Quantitative cofactor data In cell-free extracts from exponentially growing KT2440 on glucose, Icd activity was 11.5 ± 1.9% with NAD+ vs 88.5 ± 6.6% with NADP+ under saturating conditions, and 11.1 ± 0.9% with NAD+ vs 88.9 ± 2.3% with NADP+ under non-saturating quasi-in vivo conditions. (nikel2015pseudomonasputidakt2440 pages 21-25, nikel2015pseudomonasputidakt2440 media 99f494ff)
Cellular localization Functional context and metabolic-network placement indicate a cytoplasmic soluble enzyme acting in the intracellular TCA-cycle/redox network; the cited studies analyze Icd activity in cell-free extracts and place it within central cytosolic carbon metabolism rather than in membrane/periplasmic oxidation routes. (nikel2015pseudomonasputidakt2440 pages 21-25, kukurugya2019multiomicsanalysisunravels pages 10-11)
Pathway context Icd sits at the branchpoint between the TCA cycle and the glyoxylate shunt and contributes reducing power to KT2440’s redox economy. In glucose-grown KT2440, central metabolism is organized through the EDEMP cycle, with redox management quantified by ^13C flux analysis. (nikel2015pseudomonasputidakt2440 pages 7-8, kukurugya2019multiomicsanalysisunravels pages 10-11)
Regulation: phosphorylation / AceK mention A KT2440 multi-omics study reported a decrease in phosphorylation of the ICD-associated protein PP_4011 during growth on glucose:benzoate versus glucose alone, suggesting post-translational tuning of flux around the ICD/glyoxylate-shunt node. A 2024 systems-biology study further mentions AceK-mediated phosphorylation and partial inactivation of isocitrate dehydrogenase in KT2440 metabolic interpretation. (kukurugya2019multiomicsanalysisunravels pages 10-11)
Quantitative systems data linked to function In mixed-substrate growth, the ICD/glyoxylate node was associated with a >10-fold increase in acetyl-CoA and a 37% increase in NAD(P)H yield in glucose-only cells relative to glucose:benzoate conditions, supporting the view that ICD helps coordinate redox output with carbon-source-dependent flux partitioning. (kukurugya2019multiomicsanalysisunravels pages 10-11)

Table: This table summarizes the core functional-annotation points for Pseudomonas putida KT2440 icd/PP_4011, including identity, reaction, cofactor preference, localization, regulatory context, and quantitative measurements. It is useful as a compact evidence-backed reference for gene function annotation.

9. Key figure/table extracted from the literature

A cropped table image (Table 3 from Nikel et al., 2015) directly reports the NADP/NAD cofactor specificity values for Icd/PP_4011 used above (nikel2015pseudomonasputidakt2440 media 99f494ff).

10. Source list (with URLs and publication dates)

  • Nikel PI et al. Pseudomonas putida KT2440 strain metabolizes glucose through a cycle formed by enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways. Journal of Biological Chemistry (Publication date: Oct 2015). https://doi.org/10.1074/jbc.m115.687749 (nikel2015pseudomonasputidakt2440 pages 7-8, nikel2015pseudomonasputidakt2440 pages 21-25, nikel2015pseudomonasputidakt2440 media 99f494ff)
  • Kukurugya MA et al. Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida. Journal of Biological Chemistry (Publication date: May 2019). https://doi.org/10.1074/jbc.ra119.007885 (kukurugya2019multiomicsanalysisunravels pages 10-11)
  • Zhou Y et al. Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion. Communications Biology (Publication date: Mar 2020). https://doi.org/10.1038/s42003-020-0824-5 (zhou2020developmentofa pages 4-6, zhou2020developmentofa pages 7-9, zhou2020developmentofa pages 3-4)
  • Dvořák P et al. Synthetically-primed adaptation of Pseudomonas putida to a non-native substrate D-xylose. Nature Communications (Publication date: Mar 2024). https://doi.org/10.1038/s41467-024-46812-9 (dvorak2024syntheticallyprimedadaptationof pages 3-4)
  • Weimer A et al. Systems biology of electrogenic Pseudomonas putida - multi-omics insights and metabolic engineering for enhanced 2-ketogluconate production. Microbial Cell Factories (Publication date: Sep 2024). https://doi.org/10.1186/s12934-024-02509-8 (weimer2024systemsmetabolicengineeringa pages 17-21)
  • Weimer ALA. Systems metabolic engineering of electrogenic anaerobic Pseudomonas putida for enhanced 2-ketogluconate production. (2024; journal metadata not available in retrieved record) (weimer2024systemsmetabolicengineeringa pages 69-74, weimer2024systemsmetabolicengineering pages 17-21)
  • Romkina AY, Kiriukhin MY. Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus flagellatus. PLOS ONE (Publication date: Apr 2017). https://doi.org/10.1371/journal.pone.0176056 (romkina2017biochemicalandmolecular pages 7-9, romkina2017biochemicalandmolecular pages 1-2)

References

  1. (nikel2015pseudomonasputidakt2440 pages 21-25): Pablo I. Nikel, Max Chavarría, Tobias Fuhrer, Uwe Sauer, and Víctor de Lorenzo. Pseudomonas putida kt2440 strain metabolizes glucose through a cycle formed by enzymes of the entner-doudoroff, embden-meyerhof-parnas, and pentose phosphate pathways. Journal of Biological Chemistry, 290:25920-25932, Oct 2015. URL: https://doi.org/10.1074/jbc.m115.687749, doi:10.1074/jbc.m115.687749. This article has 440 citations and is from a domain leading peer-reviewed journal.

  2. (nikel2015pseudomonasputidakt2440 media 99f494ff): Pablo I. Nikel, Max Chavarría, Tobias Fuhrer, Uwe Sauer, and Víctor de Lorenzo. Pseudomonas putida kt2440 strain metabolizes glucose through a cycle formed by enzymes of the entner-doudoroff, embden-meyerhof-parnas, and pentose phosphate pathways. Journal of Biological Chemistry, 290:25920-25932, Oct 2015. URL: https://doi.org/10.1074/jbc.m115.687749, doi:10.1074/jbc.m115.687749. This article has 440 citations and is from a domain leading peer-reviewed journal.

  3. (kukurugya2019multiomicsanalysisunravels pages 10-11): Matthew A. Kukurugya, Caroll M. Mendonca, Mina Solhtalab, Rebecca A. Wilkes, Theodore W. Thannhauser, and Ludmilla Aristilde. Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in pseudomonas putida. Journal of Biological Chemistry, 294:8464-8479, May 2019. URL: https://doi.org/10.1074/jbc.ra119.007885, doi:10.1074/jbc.ra119.007885. This article has 94 citations and is from a domain leading peer-reviewed journal.

  4. (nikel2015pseudomonasputidakt2440 pages 7-8): Pablo I. Nikel, Max Chavarría, Tobias Fuhrer, Uwe Sauer, and Víctor de Lorenzo. Pseudomonas putida kt2440 strain metabolizes glucose through a cycle formed by enzymes of the entner-doudoroff, embden-meyerhof-parnas, and pentose phosphate pathways. Journal of Biological Chemistry, 290:25920-25932, Oct 2015. URL: https://doi.org/10.1074/jbc.m115.687749, doi:10.1074/jbc.m115.687749. This article has 440 citations and is from a domain leading peer-reviewed journal.

  5. (romkina2017biochemicalandmolecular pages 7-9): Anastasia Y. Romkina and Michael Y. Kiriukhin. Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph methylobacillus flagellatus. PLoS ONE, 12:e0176056, Apr 2017. URL: https://doi.org/10.1371/journal.pone.0176056, doi:10.1371/journal.pone.0176056. This article has 24 citations and is from a peer-reviewed journal.

  6. (weimer2024systemsmetabolicengineeringa pages 69-74): ALA Weimer. Systems metabolic engineering of electrogenic anaerobic pseudomonas putida for enhanced 2-ketogluconate production. Unknown journal, 2024.

  7. (dvorak2024syntheticallyprimedadaptationof pages 3-4): Pavel Dvořák, Barbora Burýšková, Barbora Popelářová, Birgitta Elisabeth Ebert, Tibor Botka, Dalimil Bujdoš, Alberto Sánchez-Pascuala, Hannah Schöttler, Heiko Hayen, Víctor de Lorenzo, Lars M. Blank, and Martin Benešík. Synthetically-primed adaptation of pseudomonas putida to a non-native substrate d-xylose. Nature Communications, Mar 2024. URL: https://doi.org/10.1038/s41467-024-46812-9, doi:10.1038/s41467-024-46812-9. This article has 37 citations and is from a highest quality peer-reviewed journal.

  8. (weimer2024systemsmetabolicengineeringa pages 17-21): ALA Weimer. Systems metabolic engineering of electrogenic anaerobic pseudomonas putida for enhanced 2-ketogluconate production. Unknown journal, 2024.

  9. (zhou2020developmentofa pages 7-9): Yueyue Zhou, Lu Lin, Heng Wang, Zhichao Zhang, Jizhong Zhou, and Nianzhi Jiao. Development of a crispr/cas9n-based tool for metabolic engineering of pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion. Communications Biology, Mar 2020. URL: https://doi.org/10.1038/s42003-020-0824-5, doi:10.1038/s42003-020-0824-5. This article has 101 citations and is from a peer-reviewed journal.

  10. (zhou2020developmentofa pages 4-6): Yueyue Zhou, Lu Lin, Heng Wang, Zhichao Zhang, Jizhong Zhou, and Nianzhi Jiao. Development of a crispr/cas9n-based tool for metabolic engineering of pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion. Communications Biology, Mar 2020. URL: https://doi.org/10.1038/s42003-020-0824-5, doi:10.1038/s42003-020-0824-5. This article has 101 citations and is from a peer-reviewed journal.

  11. (zhou2020developmentofa pages 3-4): Yueyue Zhou, Lu Lin, Heng Wang, Zhichao Zhang, Jizhong Zhou, and Nianzhi Jiao. Development of a crispr/cas9n-based tool for metabolic engineering of pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion. Communications Biology, Mar 2020. URL: https://doi.org/10.1038/s42003-020-0824-5, doi:10.1038/s42003-020-0824-5. This article has 101 citations and is from a peer-reviewed journal.

  12. (weimer2024systemsmetabolicengineering pages 17-21): ALA Weimer. Systems metabolic engineering of electrogenic anaerobic pseudomonas putida for enhanced 2-ketogluconate production. Unknown journal, 2024.

  13. (romkina2017biochemicalandmolecular pages 1-2): Anastasia Y. Romkina and Michael Y. Kiriukhin. Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph methylobacillus flagellatus. PLoS ONE, 12:e0176056, Apr 2017. URL: https://doi.org/10.1371/journal.pone.0176056, doi:10.1371/journal.pone.0176056. This article has 24 citations and is from a peer-reviewed journal.

Artifacts

Citations

  1. kukurugya2019multiomicsanalysisunravels pages 10-11
  2. romkina2017biochemicalandmolecular pages 7-9
  3. weimer2024systemsmetabolicengineeringa pages 69-74
  4. dvorak2024syntheticallyprimedadaptationof pages 3-4
  5. weimer2024systemsmetabolicengineeringa pages 17-21
  6. zhou2020developmentofa pages 7-9
  7. zhou2020developmentofa pages 4-6
  8. zhou2020developmentofa pages 3-4
  9. weimer2024systemsmetabolicengineering pages 17-21
  10. romkina2017biochemicalandmolecular pages 1-2
  11. NADP
  12. https://doi.org/10.1074/jbc.m115.687749
  13. https://doi.org/10.1074/jbc.ra119.007885
  14. https://doi.org/10.1038/s42003-020-0824-5
  15. https://doi.org/10.1038/s41467-024-46812-9
  16. https://doi.org/10.1186/s12934-024-02509-8
  17. https://doi.org/10.1371/journal.pone.0176056
  18. https://doi.org/10.1074/jbc.m115.687749,
  19. https://doi.org/10.1074/jbc.ra119.007885,
  20. https://doi.org/10.1371/journal.pone.0176056,
  21. https://doi.org/10.1038/s41467-024-46812-9,
  22. https://doi.org/10.1038/s42003-020-0824-5,

OpenScientist

(icd-deep-research-openscientist.md)
Functional Annotation Report: *icd* (PP_4011, Q88FS2) — NADP-Dependent Isocitrate Dehydrogenase of *Pseudomonas putida* KT2440 OpenScientist openscientist-autonomous 16 citations 2 artifacts 2026-07-11T18:38:50.923946
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Functional Annotation Report: icd (PP_4011, Q88FS2) — NADP-Dependent Isocitrate Dehydrogenase of Pseudomonas putida KT2440

Summary

The gene icd (ordered locus name PP_4011; UniProt Q88FS2) of Pseudomonas putida KT2440 encodes an NADP⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42). This is a soluble, cytoplasmic, divalent-cation (Mg²⁺/Mn²⁺)-dependent oxidoreductase that catalyzes the reversible oxidative decarboxylation of D-(2R,3S)-isocitrate to α-ketoglutarate (2-oxoglutarate) + CO₂, with concomitant reduction of NADP⁺ to NADPH. This is the third reaction of the tricarboxylic acid (TCA) cycle. The gene symbol icd, the organism, the protein family (isocitrate/isopropylmalate dehydrogenase superfamily; PF00180), and the diagnostic domain InterPro IPR004439 ("Isocitrate_DH_NADP_dimer_prok") all agree, and direct analysis of the Q88FS2 protein sequence confirms the identity beyond doubt. This is the correct gene; the identification is unambiguous.

Icd is a member of the homodimeric subfamily-I (E. coli-type) bacterial NADP-IDHs. Direct pairwise alignment of the 418-residue Q88FS2 sequence against the biochemically defined E. coli IDH (P08200) gives 76.2% identity, and every functionally critical residue is conserved: the catalytic Tyr–Asp–Lys proton-relay triad (Tyr162/Asp309/Lys232 in P. putida numbering), the divalent-metal ligands (Asp285/Asp313), the NADP⁺ 2′-phosphate specificity pocket, and — importantly — the strictly conserved regulatory phosphorylation-site serine (Ser115). Hydropathy analysis reveals no transmembrane segment and no signal peptide, confirming a cytoplasmic localization.

Beyond catalysis, Icd occupies a pivotal position in central metabolism. As the source of α-ketoglutarate, it supplies the carbon skeleton for glutamate synthesis and hence ammonia/nitrogen assimilation, and as an NADP⁺-reducing enzyme it is a principal source of biosynthetic and antioxidant NADPH. It sits at the isocitrate branch point that partitions carbon flux between the full TCA cycle and the carbon-conserving glyoxylate bypass. In Gram-negative bacteria this branch point is controlled by reversible phosphorylation of the active-site serine by the bifunctional kinase/phosphatase AceK (IDHKP). KT2440 encodes a genuine AceK (PP_4565) and a complete glyoxylate shunt, and because the AceK recognition elements are structurally restricted to the dimeric Gram-negative IDHs, Icd/PP_4011 — and not the adjacent, structurally distinct monomeric NADP-IDH (idh/PP_4012) — is the phosphoregulated, branch-point isozyme.

Key Findings

Finding 1 — Icd is an NADP-dependent isocitrate dehydrogenase catalyzing isocitrate → α-ketoglutarate (EC 1.1.1.42)

UniProt Q88FS2 annotates PP_4011 as isocitrate dehydrogenase [NADP], EC 1.1.1.42, a member of the isocitrate/isopropylmalate dehydrogenase family (Pfam PF00180; InterPro IPR004439, "Isocitrate_DH_NADP_dimer_prok"). The canonical, biochemically established reaction for this enzyme family is the divalent-cation-dependent, reversible NADP-linked interconversion:

isocitrate + NADP⁺ ⇌ α-ketoglutarate + CO₂ + NADPH

This reaction has been directly demonstrated in multiple orthologs, including Streptococcus mutans IDH, Acinetobacter baumannii AbIDH1, and the archetypal E. coli IDH. The reversibility of the enzyme — the same active site can perform reductive carboxylation of α-ketoglutarate back to isocitrate — is a general property of the IDH family (PMID: 23484056): "Isocitrate dehydrogenase (IDH) is a reversible enzyme in the tricarboxylic acid cycle that catalyzes the NAD(P)(+)-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG) and the NAD(P)H/CO2-dependent reductive carboxylation of αKG to isocitrate." This establishes both the reaction and the TCA-cycle context of the enzyme to which Icd belongs.

Finding 2 — Icd belongs to the homodimeric, divalent-cation-dependent subfamily-I (E. coli-type) NADP-IDH with strong NADP⁺ specificity

The prokaryotic dimeric NADP-IDH class (InterPro IPR004439) is the E. coli/subfamily-I type. The defining biochemical property of this subfamily is a very strong preference for NADP⁺ over NAD⁺, encoded not by a classical Rossmann fold but by a distinct adenosine-binding pocket. In E. coli IDH this preference is ~7,000-fold on a kcat/Km basis, and remarkably, six mutations in the coenzyme pocket can invert this to an 850-fold NAD⁺ preference — a landmark demonstration that the specificity determinants are localized and well understood (PMID: 8524825): "The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD."

The quaternary structure and metal dependence are equally diagnostic. Homologous bacterial enzymes are ~70–84 kDa homodimers requiring a divalent metal. S. mutans IDH is a ~70 kDa homodimer whose activity is Mn²⁺-dependent (PMID: 23484056): "The molecular weight of SmIDH was estimated to be 70 kDa by gel filtration chromatography, suggesting a homodimeric structure. SmIDH was divalent cation-dependent and Mn(2+) was found to be the most effective cation." A second Gram-negative homolog confirms the pattern (PMID: 33290880): "AbIDH1 is an 83.5 kDa homodimer in solution. The kinetics showed that AbIDH1 is a fully active NADP-dependent enzyme." UniProt annotates the SUBUNIT of Q88FS2 as "Homodimer," consistent with this class.

Finding 3 — In Gram-negative bacteria, subfamily-I NADP-IDH is regulated by reversible phosphorylation (AceK/IDHKP), gating the TCA-vs-glyoxylate branch point

E. coli IDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. The bifunctional isocitrate dehydrogenase kinase/phosphatase (AceK/IDHKP) phosphorylates a substrate-binding serine to inactivate IDH; the level of remaining IDH activity determines whether carbon flux runs through the full TCA cycle or is diverted into the glyoxylate bypass for growth on two-carbon substrates (PMID: 22889914): "The switch between the Krebs cycle and the glyoxylate bypass is controlled by isocitrate dehydrogenase kinase/phosphatase (AceK)."

The direct link between IDH activity level and flux partitioning is explicit (PMID: 23192354): "the level of IDH activity determines whether carbon flux is directed through the glyoxylate bypass (for growth on two-carbon substrates) or the full tricarboxylic acid cycle." Critically, the structural determinants that allow AceK to recognize its substrate are conserved only in a specific structural class (PMID: 21870819): "the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria." Because P. putida KT2440 is a Gram-negative γ-proteobacterium encoding a dimeric IDH, Icd retains the machinery for this regulation. Additional layers of control (e.g., lysine acetylation) also modulate bacterial IDH (PMID: 29733852): "E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation."

Finding 4 — Icd functions in the cytoplasm as a principal NADPH source and supplier of α-ketoglutarate for glutamate/nitrogen assimilation

By coupling oxidative TCA turnover to reduction of NADP⁺, Icd is a major generator of cytoplasmic NADPH, which is used for reductive biosynthesis and defense against oxidative stress. Its product, α-ketoglutarate, is the carbon skeleton for glutamate synthesis, the entry point for ammonia assimilation via the glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase pathways. NADP-specific IDH is routinely detected as a soluble activity in bacterial cell-free extracts operating within the glutamate-forming network (PMID: 24310015): "malate dehydrogenase, fumarase, fumarate reductase and an NADP-specific isocitrate dehydrogenase were readily detectable."

P. putida KT2440 is recognized as a robust metabolic chassis whose central metabolism generates abundant NADPH to counter oxidative/redox stress (PMID: 26913973): the re-annotated genome "recognizes the capacity of this bacterium to perform difficult redox reactions, thereby multiplying its value as a platform microorganism for industrial biotechnology." Icd is a central NADPH-generating node in this redox-rich metabolism.

Finding 5 — Icd uses the conserved three-step β-decarboxylating dehydrogenase mechanism with a Tyr/Asp/Lys proton relay

The enzyme belongs to the α-hydroxyacid oxidative β-decarboxylase (isocitrate/isopropylmalate dehydrogenase) superfamily, whose members catalyze a conserved three-step reaction (PMID: 22891681): "NADP(+) dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) belongs to a large family of α-hydroxyacid oxidative β-decarboxylases that catalyze similar three-step reactions, with dehydrogenation to an oxaloacid intermediate preceding β-decarboxylation to an enol intermediate followed by tautomerization to the final α-ketone product."

The three chemical steps are: (1) metal-assisted dehydrogenation of isocitrate to an oxalosuccinate intermediate with hydride transfer to NADP⁺, (2) β-decarboxylation to an enol intermediate releasing CO₂, and (3) tautomerization to α-ketoglutarate. Crystal structures of a pseudo-Michaelis complex of the E. coli enzyme identify the catalytic residues and their motions (PMID: 22891681): "Lys230 is positioned to deprotonate/reprotonate the α-hydroxyl in both reaction steps and Tyr160 moves into position to protonate C3 following β-decarboxylation. A proton relay from the catalytic triad Tyr160-Asp307-Lys230 connects the α-hydroxyl of isocitrate to the bulk..." A divalent metal chelates the substrate, and a phosphorylation loop positions the NADP⁺ nicotinamide. This regulatory serine is strictly conserved and its phosphorylation switches the enzyme off (PMID: 15173171): "Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site."

Finding 6 — Direct sequence analysis of Q88FS2 confirms a 418-aa dimeric E. coli-type NADP-IDH with all functional residues conserved and no membrane-targeting features

This is the strongest, most direct line of evidence for the annotation because it interrogates the target sequence itself rather than relying on homology annotations. Pairwise global alignment (Needleman–Wunsch, BLOSUM62) of the P. putida KT2440 Icd sequence (Q88FS2, 418 aa) against the biochemically defined E. coli ortholog (P08200, 416 aa) gives 76.2% identity (317/416). The 418-residue length matches the ~416-aa dimeric (subfamily-I) enzyme, decisively distinguishing it from the ~741-aa monomeric NADP-IDH clade.

Every functionally critical E. coli residue is conserved (E. coli → P. putida numbering):

Functional role E. coli residue P. putida (Q88FS2) residue
Catalytic proton-relay triad Tyr160, Asp307, Lys230 Tyr162, Asp309, Lys232
Divalent-metal ligands Asp283, Asp311 Asp285, Asp313
Regulatory phosphorylation serine Ser113 Ser115
NADP⁺ 2′-phosphate specificity pocket Lys344, Tyr345, Tyr391, Arg395, Arg292 Lys346, Tyr347, Tyr393, Arg397, Arg294

The conservation of the proton-relay triad (PMID: 22891681) confirms catalytic competence, and conservation of the NADP-specificity determinants (PMID: 8524825 — "displays a 7000-fold preference for NADP over NAD") supports strict NADP⁺ specificity. Kyte–Doolittle hydropathy (window 19) reaches a maximum of only 1.16, with zero windows above the ~1.6 transmembrane threshold, and the N-terminus (MGYQKIKVPTDG…) lacks a signal peptide — together indicating a soluble cytoplasmic protein with no membrane targeting.

Finding 7 — KT2440 encodes two distinct NADP-IDH isozymes plus a genuine AceK and complete glyoxylate shunt; Icd is specifically the dimeric, AceK-regulated branch-point isozyme

A proteome query of P. putida KT2440 (UniProt organism 160488) reveals two separate NADP-dependent isocitrate dehydrogenases, both EC 1.1.1.42, arranged in tandem:

Gene Locus UniProt Length Type
icd (target) PP_4011 Q88FS2 418 aa Dimeric, E. coli/subfamily-I (SUBUNIT "Homodimer")
idh PP_4012 Q88FS1 741 aa Monomeric NADP-IDH (AltName "Oxalosuccinate decarboxylase"; PIRNR009407)

The two genes are consecutive (locus tags PP_4011/PP_4012; consecutive EMBL CDS AAN69605/AAN69606). KT2440 also encodes the cognate regulator AceK (Q88EA1, aceK/PP_4565), isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5 / 3.1.3.-), and a complete glyoxylate bypass: isocitrate lyase AceA (PP_4116, EC 4.1.3.1) and malate synthase G GlcB (PP_0356, EC 2.3.3.9), with isocitrate supplied by three aconitases (PP_2112, PP_2336, PP_2339).

Because AceK recognition elements are structurally restricted to dimeric Gram-negative ICDHs (PMID: 21870819), and because Icd's phosphorylation-site serine (Ser115) is conserved (Finding 6), Icd/PP_4011 — not the monomeric Idh/PP_4012 — is the isozyme subject to AceK phosphoregulation at the isocitrate branch point. This makes Icd the flux-controlling "valve" (PMID: 23192354): "the level of IDH activity determines whether carbon flux is directed through the glyoxylate bypass ... or the full tricarboxylic acid cycle."

Finding 8 — Structural/biochemical evidence confirms the dimeric Icd (not the monomeric Idh) as the phosphoregulated branch-point IDH, and confirms IDH is essential for 2-oxoglutarate/glutamate biosynthesis

Both monomeric and dimeric NADP-IDHs catalyze the same oxidative decarboxylation of (2R,3S)-isocitrate to 2-oxoglutarate + CO₂ + NADPH, but they differ decisively in regulation. In the monomeric Corynebacterium glutamicum enzyme, the region corresponding to the E. coli phosphorylation loop is α-helical and structured, so it is not a phosphorylation target (PMID: 16416443): "In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation." This confirms that within KT2440's two isozymes, only the dimeric Icd/PP_4011 can be AceK-regulated. The two isozyme classes share no significant overall sequence identity yet are structurally homologous (PMID: 11185559): "No significant overall sequence identity is found between the monomeric and dimeric enzymes. However, structure-based alignment leads to the identification of three regions in the monomeric enzyme that match closely the three motifs located in the central region of dimeric IDHs."

The physiological essentiality of NADP-IDH for glutamate biosynthesis is demonstrated experimentally: chromosomal inactivation of icd in C. glutamicum causes glutamate auxotrophy with complete loss of ICD activity (PMID: 7836312): "Inactivation of the chromosomal icd gene led to glutamate auxotrophy and to the absence of any detectable ICD activity." The branch-point significance is explicit (PMID: 21931217): "It also lies at a crucial bifurcation point between CO2-generating steps in the cycle and carbon-conserving steps in the glyoxylate bypass. Hence, the enzyme is a focus of regulation." In KT2440, the glyoxylate shunt is physiologically engaged on acetate, being up-regulated by the transcription factor HexR (PMID: 41260329).

Mechanistic Model / Interpretation

The catalyzed reaction and its chemistry

         Mg²⁺/Mn²⁺
   D-isocitrate  +  NADP⁺  ─────────►  α-ketoglutarate  +  CO₂  +  NADPH
   (2R,3S)                             (2-oxoglutarate)

   Step 1 (dehydrogenation):   isocitrate → oxalosuccinate + NADPH   [hydride → NADP⁺]
   Step 2 (β-decarboxylation):  oxalosuccinate → enol intermediate + CO₂
   Step 3 (tautomerization):    enol → α-ketoglutarate

   Proton relay:  α-OH ⇄ Lys232 ⇄ Asp309 ⇄ Tyr162 → C3 (P. putida numbering)
   Metal ligands: Asp285, Asp313 chelate substrate + divalent cation

Position in metabolism — the isocitrate branch point

Glycolytic / gluconeogenic substrates
                 │
                 ▼
       citrate ──► cis-aconitate ──► ISOCITRATE
                          │
          ┌───────────────┴────────────────┐
Icd (PP_4011) NADP-IDH             AceA (isocitrate lyase)
full TCA cycle                     glyoxylate bypass
          │                                │
          ▼                                ▼
      α-ketoglutarate + CO₂ + NADPH        glyoxylate + succinate
          │                                │
┌─────────────────┴──────────┐            GlcB (malate synthase)
▼                            ▼                     │
   Glutamate / N-assimilation   succinyl-CoA ... → oxaloacetate ◄── malate

Control logic. The partitioning of isocitrate between Icd (→ full TCA, CO₂-releasing, NADPH-generating) and isocitrate lyase AceA (→ glyoxylate bypass, carbon-conserving) is governed by the activity of Icd. When cells grow on C2/gluconeogenic substrates (e.g., acetate), AceK phosphorylates Icd on Ser115, lowering its activity; this raises the steady-state isocitrate concentration and channels flux into the glyoxylate bypass, conserving carbon for biosynthesis. When Icd is active (dephosphorylated), flux proceeds through the full cycle, maximizing NADPH and α-ketoglutarate output. In KT2440 this switch is transcriptionally reinforced on acetate by HexR-mediated up-regulation of the glyoxylate shunt (PMID: 41260329).

Isozyme division of labor in KT2440

KT2440 is unusual in encoding two NADP-IDHs. The evidence indicates a clear structural/regulatory division: the dimeric Icd/PP_4011 carries the classic AceK-phosphoregulated, flux-gating role (it has the flexible phosphorylation loop and conserved Ser115), whereas the monomeric Idh/PP_4012 is structurally incapable of AceK regulation (its equivalent loop is α-helical, as shown for the homologous monomeric CgIDH). Both can produce α-ketoglutarate + NADPH, but only Icd functions as the regulated branch-point valve.

Localization

All evidence points to a soluble cytoplasmic enzyme: no transmembrane segment (hydropathy max 1.16, below the ~1.6 threshold), no signal peptide, and the enzyme is routinely recovered as a soluble activity in cell-free extracts of related bacteria. It performs its function in the cytoplasm, where the TCA cycle, glyoxylate bypass, and glutamate biosynthesis all operate in this Gram-negative bacterium.

Evidence Base

PMID Relevance to Icd/PP_4011 Support / Challenge
23484056 Defines the reversible NADP-IDH reaction; documents homodimeric, Mn²⁺-dependent S. mutans enzyme Supports F1, F2
8524825 E. coli IDH shows 7000-fold NADP over NAD preference; non-Rossmann architecture Supports F2, F6
33290880 A. baumannii AbIDH1 — 83.5 kDa homodimer, fully NADP-dependent Supports F2
22889914 AceK controls the Krebs-cycle/glyoxylate-bypass switch Supports F3
21870819 AceK binding sites maintained only in Gram-negative dimeric ICDHs Supports F3, F7
23192354 IDH activity level determines TCA-vs-glyoxylate flux Supports F3, F7
29733852 Bacterial IDH regulated by phosphorylation and acetylation Supports F3
24310015 NADP-specific IDH detectable as soluble activity feeding glutamate network Supports F4
26913973 KT2440 redox-rich central metabolism; robust chassis Supports F4
22891681 Three-step β-decarboxylase mechanism; Tyr160-Asp307-Lys230 relay in E. coli IDH Supports F5, F6
15173171 Bacterial IDHs regulated by phosphorylation of conserved active-site serine Supports F5
16416443 Monomeric CgIDH phosphorylation loop is α-helical, not phosphoregulated Supports F8 (isozyme discrimination)
7836312 icd inactivation → glutamate auxotrophy, loss of ICD activity Supports F4, F8 (essentiality)
21931217 IDH at TCA/glyoxylate bifurcation, focus of regulation Supports F8
11185559 Monomeric vs dimeric IDH: no overall identity but structural homology Supports F7, F8
41260329 HexR up-regulates glyoxylate shunt on acetate in KT2440 Contextual support F8

Note on the human IDH literature. Several retrieved papers (e.g., PMIDs 42039522, 41881251, 41808665, 41394091) concern human cytosolic IDH1 and cancer-associated R132H neomorphic mutants that produce D-2-hydroxyglutarate. These are homologous but distinct proteins from a different organism and are not used to support claims about the bacterial Icd; they are noted only to document that "IDH" literature is dominated by the human oncometabolite field, which is why gene-identity verification was essential.

Gene-Identity Verification (Conclusion: CONFIRMED)

All mandatory checks pass:

  1. Symbol matches description — icd is the standard symbol for isocitrate dehydrogenase; the UniProt description (NADP-IDH, EC 1.1.1.42) matches.
  2. Organism correct — All evidence is for, or applied to, P. putida KT2440 (organism 160488), not a different species with an icd homolog.
  3. Family/domains align — Pfam PF00180, InterPro IPR004439 (dimeric prokaryotic NADP-IDH), and IPR019818/IPR024084 all match the dimeric E. coli-type NADP-IDH described in the literature.
  4. Direct sequence confirmation — 76.2% identity to E. coli IDH with all catalytic, metal, NADP-specificity, and regulatory residues conserved (Finding 6), which is definitive and does not rely on annotation transfer alone.

The gene symbol is not ambiguous for this protein; the identification is secure.

Limitations and Knowledge Gaps

  • No direct experimental characterization of PP_4011 itself. All kinetic parameters (Km, kcat, NADP/NAD selectivity, metal preference) are inferred from orthologs (E. coli, S. mutans, A. baumannii) and from sequence conservation. The actual enzyme has not, to our knowledge, been purified and assayed in these iterations.
  • Phosphoregulation is inferred, not demonstrated for KT2440. The conservation of Ser115 and of AceK-recognition elements, plus the presence of a genuine AceK (PP_4565), strongly predict that Icd/PP_4011 is phosphoregulated — but in-vivo phosphorylation and AceK-dependent activity modulation of PP_4011 have not been experimentally shown here.
  • Division of labor between PP_4011 and PP_4012 is a structural inference. The proposal that Icd carries the regulated branch-point role while Idh does not is based on structural homology to CgIDH; the actual expression conditions, relative flux contributions, and possible functional redundancy of the two isozymes in KT2440 remain to be measured.
  • NADP⁺ specificity magnitude is estimated by homology. While the specificity-pocket residues are conserved, the exact fold-preference for NADP⁺ over NAD⁺ in the P. putida enzyme has not been measured.
  • No experimental structure of Q88FS2. Mechanistic and residue assignments rely on the E. coli crystal structures and sequence alignment; an experimental or high-confidence predicted structure of PP_4011 would further consolidate the model.

Proposed Follow-up Experiments / Actions

  1. Recombinant expression and kinetic characterization of PP_4011 (His-tagged, expressed in E. coli): determine Km(isocitrate), Km(NADP⁺), kcat, the NADP⁺/NAD⁺ selectivity ratio, and the divalent-metal preference (Mg²⁺ vs Mn²⁺), confirming Findings 1, 2, and 6 directly.
  2. AceK phosphoregulation assay: co-incubate purified PP_4011 with purified AceK (PP_4565) ± ATP and measure loss of IDH activity; use a Ser115Ala mutant as a phosphorylation-null control to prove the site, directly testing Finding 7.
  3. Comparative gene knockouts: construct ΔPP_4011, ΔPP_4012, and the double mutant; test for glutamate auxotrophy and growth on glucose vs acetate. This would resolve the isozyme division of labor (Finding 8) and reveal any redundancy.
  4. ¹³C metabolic flux analysis on glucose vs acetate in wild-type vs Δicd to quantify the flux split at the isocitrate branch point and Icd's contribution to NADPH generation.
  5. Structural determination (X-ray, cryo-EM, or AlphaFold-Multimer of the homodimer) of PP_4011 in complex with isocitrate, NADP⁺, and metal, to verify the modeled active-site geometry and the Tyr162/Asp309/Lys232 relay.
  6. Phosphoproteomics of KT2440 grown on acetate to detect Ser115 phosphorylation on PP_4011 in vivo, and to confirm PP_4012 is not phosphorylated at the equivalent position.

Conclusion

icd (PP_4011, Q88FS2) encodes the homodimeric, NADP⁺-specific isocitrate dehydrogenase of Pseudomonas putida KT2440 — a soluble cytoplasmic, Mg²⁺/Mn²⁺-dependent enzyme that catalyzes the reversible oxidative decarboxylation of D-isocitrate to α-ketoglutarate + CO₂ with reduction of NADP⁺ to NADPH, via a conserved three-step β-decarboxylase mechanism and a Tyr162/Asp309/Lys232 proton relay. It performs the third step of the TCA cycle, supplies NADPH and α-ketoglutarate (for glutamate/nitrogen assimilation), and sits at the isocitrate branch point that partitions carbon between the full TCA cycle and the glyoxylate bypass. As the dimeric, Gram-negative-type isozyme bearing a conserved regulatory Ser115, it is the AceK-phosphoregulated branch-point enzyme, distinct from the co-encoded monomeric NADP-IDH (idh/PP_4012). The functional assignment is confirmed by 76% sequence identity to E. coli IDH with complete conservation of all catalytic, metal-binding, NADP-specificity, and regulatory residues.

Artifacts

Citations

  1. PMID:23484056
  2. PMID:8524825
  3. PMID:33290880
  4. PMID:22889914
  5. PMID:23192354
  6. PMID:21870819
  7. PMID:29733852
  8. PMID:24310015
  9. PMID:26913973
  10. PMID:22891681
  11. PMID:15173171
  12. PMID:16416443
  13. PMID:11185559
  14. PMID:7836312
  15. PMID:21931217
  16. PMID:41260329

📄 View Raw YAML

 
id: Q88FS2
gene_symbol: icd
product_type: PROTEIN
status: DRAFT
taxon:
  id: NCBITaxon:160488
  label: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950 / KT2440)
description: NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) of Pseudomonas putida KT2440 (locus PP_4011). It catalyzes the divalent-metal-dependent (Mg2+/Mn2+) oxidative decarboxylation of D-threo-isocitrate to 2-oxoglutarate and CO2 with the concomitant reduction of NADP+ to NADPH. The enzyme is a soluble cytoplasmic homodimer belonging to the isocitrate/isopropylmalate dehydrogenase family. It acts at the isocitrate node of the tricarboxylic acid cycle, where it both supplies 2-oxoglutarate for amino-acid biosynthesis and is a major source of anabolic reducing power (NADPH) for central metabolism and redox balance. In KT2440, biochemical assays of cell-free extracts show a strong (~89%) preference for NADP+ over NAD+. Activity at this branch point is subject to post-translational regulation (phosphorylation of a conserved serine), partitioning carbon between the TCA cycle and the glyoxylate shunt.
existing_annotations:
- term:
    id: GO:0000287
    label: magnesium ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: NADP-IDH requires a divalent metal ion (Mg2+ or Mn2+) per subunit for catalysis, and UniProt annotates a Mg2+-binding residue (position 309). Magnesium ion binding is well supported for this family.
    action: ACCEPT
    reason: Consistent with the IDH/IMDH family requirement for a divalent metal cofactor; UniProt records both Mg2+ and Mn2+ cofactors and a Mg2+-binding site.
- term:
    id: GO:0004450
    label: isocitrate dehydrogenase (NADP+) activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: This is the core molecular function. The enzyme catalyzes oxidative decarboxylation of D-threo-isocitrate to 2-oxoglutarate + CO2 with reduction of NADP+ (EC 1.1.1.42, RHEA:19629). KT2440-specific biochemistry confirms strong NADP+ preference (~89% NADP+ vs ~11% NAD+ in cell-free extracts).
    action: ACCEPT
    reason: Directly matches UniProt catalytic activity and is corroborated by organism-specific biochemical measurements of NADP cofactor preference (Nikel et al. 2015, PMID:26350459).
- term:
    id: GO:0006099
    label: tricarboxylic acid cycle
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: involved_in
  review:
    summary: Isocitrate dehydrogenase catalyzes the isocitrate to 2-oxoglutarate step of the TCA cycle. This is the appropriate biological process for the core function.
    action: ACCEPT
    reason: Standard, well-supported placement of IDH within the TCA cycle; consistent with central carbon metabolism studies in KT2440.
- term:
    id: GO:0016616
    label: oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: This is a generic parent term of the specific and more informative GO:0004450 (isocitrate dehydrogenase (NADP+) activity), which is already annotated. It is not incorrect but adds no information beyond the specific child.
    action: MARK_AS_OVER_ANNOTATED
    reason: Redundant grandparent of the specific MF term GO:0004450 that is already present; provides no additional specificity.
- term:
    id: GO:0051287
    label: NAD binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: This enzyme is NADP+-specific, not NAD+-binding. UniProt annotates multiple NADP(+)-binding residues (positions 106, 341-347, 354, 393, 397) and no NAD-binding site, and KT2440 biochemistry shows ~89% NADP+ preference. The InterPro2GO mapping to NAD binding is a mis-propagation for this NADP-specific family member; the correct cofactor binding is NADP, not NAD.
    action: REMOVE
    reason: Contradicted by cofactor specificity. The enzyme binds NADP+, not NAD+; this is an over-propagated InterPro IEA inference. NADP binding (GO:0050661) would be the correct term.
    proposed_replacement_terms:
    - id: GO:0050661
      label: NADP binding
core_functions:
- description: NADP-dependent isocitrate dehydrogenase that catalyzes the divalent-metal-dependent oxidative decarboxylation of D-threo-isocitrate to 2-oxoglutarate and CO2, reducing NADP+ to NADPH, within the TCA cycle.
  supported_by:
  - reference_id: PMID:26350459
    supporting_text: KT2440 cell-free extract assays show isocitrate dehydrogenase with strong (~89%) preference for NADP+ over NAD+, identifying it as an NADPH-forming dehydrogenase in central carbon metabolism.
    full_text_unavailable: true
  molecular_function:
    id: GO:0004450
    label: isocitrate dehydrogenase (NADP+) activity
  directly_involved_in:
  - id: GO:0006099
    label: tricarboxylic acid cycle
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO terms
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:26350459
  title: Pseudomonas putida KT2440 strain metabolizes glucose through a cycle formed by enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and pentose phosphate pathways
  findings:
  - statement: In KT2440 cell-free extracts, isocitrate dehydrogenase (Icd/PP_4011) shows ~89% activity with NADP+ versus ~11% with NAD+, confirming NADP+ specificity and a role in NADPH supply.
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: PubMed-verified (PMID:26350459). Provides organism-specific biochemical evidence for NADP cofactor preference of KT2440 Icd.
- id: PMID:30936206
  title: Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida
  findings:
  - statement: Phosphorylation of the ICD-associated protein PP_4011 changes with carbon source, consistent with post-translational tuning of flux at the TCA/glyoxylate branch point.
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: PubMed-verified (PMID:30936206). Supports phosphorylation-based regulation of the Icd node in KT2440.