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this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.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: sucA (PP_4189; UniProt Q88FA9) in Pseudomonas putida KT2440

0) Target verification (critical disambiguation)

Multiple KT2440-focused studies explicitly identify sucA as PP_4189 and annotate it as the α-ketoglutarate/2‑oxoglutarate dehydrogenase E1 component (SucA) in the tricarboxylic acid (TCA) cycle, matching the UniProt Q88FA9 description (2‑oxoglutarate dehydrogenase E1; EC 1.2.4.2). (avendano2023productionofselenium pages 5-6, ainelo2019pseudomonasputidaresponds pages 5-9)

1) Key concepts & definitions (current understanding)

1.1 The enzyme and the reaction catalyzed

SucA is the E1 (decarboxylase) component of the bacterial 2‑oxoglutarate dehydrogenase complex (also termed α‑ketoglutarate dehydrogenase complex; OGDHc/KGDH). (bunik2013translatingenzymologyinto pages 2-3, chakraborty2022engineeringthe2oxoglutarate pages 2-3)

At the pathway level, the complex catalyzes the oxidative decarboxylation of 2‑oxoglutarate (α‑ketoglutarate) to succinyl‑CoA:

• Overall reaction: 2‑oxoglutarate + CoA + NAD+ → succinyl‑CoA + CO2 + NADH (chakraborty2022engineeringthe2oxoglutarate pages 2-3)

This step is irreversible and is a major branch point linking carbon metabolism with nitrogen (via 2‑oxoglutarate/glutamate) and redox metabolism through NADH generation. (bunik2013translatingenzymologyinto pages 1-2)

1.2 Complex architecture, cofactors, and substrate channeling

The bacterial complex is a multienzyme assembly of:
- E1o (SucA): ThDP/TPP-dependent 2‑oxoglutarate dehydrogenase (decarboxylase) (bunik2013translatingenzymologyinto pages 2-3)
- E2o: dihydrolipoamide succinyltransferase (forms succinyl‑CoA from CoA) (bunik2013translatingenzymologyinto pages 2-3)
- E3: dihydrolipoamide dehydrogenase (FAD/NAD+-dependent reoxidation of reduced lipoamide, producing NADH) (bunik2013translatingenzymologyinto pages 2-3)

Key cofactors include ThDP (TPP), lipoate (lipoyllysine swinging arm on E2), CoA, FAD, and NAD+. (bunik2013translatingenzymologyinto pages 2-3)

Structurally, in E. coli (a reference bacterial system), the complex is described as having a 24‑mer E2 core with E1 and E3 peripherally associated, and catalysis involves transfer of intermediates via a lipoyl “swinging arm” mechanism. (chakraborty2022engineeringthe2oxoglutarate pages 2-3)

1.3 Substrate specificity and mechanistic determinants (inference relevant to annotation)

Mechanistic/structural work across bacterial systems indicates that E1o substrate specificity for 2‑oxoglutarate is supported by conserved active-site features, including a positively charged loop (e.g., Arg505-containing loop) implicated in recognizing the 5‑carboxylate of 2‑oxoglutarate. (bunik2013translatingenzymologyinto pages 3-4, bunik2013translatingenzymologyinto pages 4-6)

Engineering studies show that mutating specific E1 residues can broaden or alter substrate recognition (e.g., variants accepting substrates lacking the 5‑carboxyl group), illustrating how E1 chemistry can be retuned for nonnative substrates in related systems. (chakraborty2022engineeringthe2oxoglutarate pages 1-2)

2) sucA in Pseudomonas putida KT2440: function, pathway role, and likely localization

2.1 Core biological role in KT2440

In KT2440, sucA (PP_4189) is repeatedly described as a key Krebs/TCA cycle enzyme, consistent with the canonical OGDHc role in producing succinyl‑CoA and NADH from 2‑oxoglutarate. (avendano2023productionofselenium pages 5-6, ainelo2019pseudomonasputidaresponds pages 5-9)

2.2 Genomic/operon context and complex partners (KT2440-specific evidence)

RNA-level investigations in P. putida KT2440 place sucA in operon context with other TCA genes, including sucB and lpdG (as part of the α‑ketoglutarate dehydrogenase complex components), and suggest larger transcriptional units that can include sdh and suc genes. (geiger2019investigationofrnabased pages 18-22)

2.3 Cellular localization

The OGDHc is a soluble cytosolic enzyme complex in bacteria (as part of central carbon metabolism); consistent with this, KT2440 studies treat SucA as a central metabolic enzyme measured in whole-cell proteomes. (ainelo2019pseudomonasputidaresponds pages 5-9, kratzl2024pseudomonasputidaas pages 7-9)

3) Regulation and control: what is known and what is still uncertain

3.1 Protein abundance changes under stress (experimental evidence)

A proteomics/metabolomics study of the GraT toxin system in P. putida (KT2440-derivative background) reported that TCA-cycle enzymes including SucA are downregulated when GraT is active (ΔgraA). In extracted quantitative data, SucA shows a fold change of −2.12 (ΔgraA vs WT, 25 °C), with other enzymes in the same TCA segment also down (Idh −2.87; SucD −2.52). (ainelo2019pseudomonasputidaresponds media c323b5f8, ainelo2019pseudomonasputidaresponds pages 5-9)

These changes were interpreted as part of a coordinated decrease in flux through the isocitrate→succinate segment of the TCA cycle. (ainelo2019pseudomonasputidaresponds pages 5-9)

3.2 RNA-based regulation (KT2440 and broader Pseudomonadaceae context)

Work on RNA motifs reports a conserved sucA RNA / sucA‑II motif in the 5′ UTR and notes two transcription start sites in P. putida KT2440, including one ~110 nt upstream of the ORF. The motif was proposed as a potential cis-regulatory element because predicted structures overlap the Shine–Dalgarno region. (geiger2019investigationofrnabased pages 22-27, geiger2019investigationofrnabased pages 83-87)

However, extensive ligand screening and follow-up experiments did not validate a classic small-molecule riboswitch mechanism for sucA‑II. Instead, results suggested possible protein-mediated regulation; RNA pulldown experiments nominated candidate interacting proteins including SucC/SucD (consistent with potential operon-level feedback). (geiger2019investigationofrnabaseda pages 83-87, geiger2019investigationofrnabased pages 83-87)

Overall, the best-supported current interpretation from these data is that post-transcriptional control may exist, but its mechanism and physiological ligand(s) remain unresolved. (geiger2019investigationofrnabased pages 83-87, geiger2019investigationofrnabaseda pages 83-87)

4) Recent developments (prioritizing 2023–2024)

4.1 2023: sucA links central metabolism to selenium nanoparticle biogenesis

A 2023 Microbial Biotechnology study of aerobic selenite reduction in P. putida KT2440 used genetic approaches and identified sucA (PP_4189) among genes implicating 2‑ketoglutarate/glutamate metabolism as important for converting selenite to selenium. The authors explicitly identify sucA as encoding 2‑oxoglutarate dehydrogenase and a key Krebs cycle enzyme. (avendano2023productionofselenium pages 5-6)

Quantitative/experimental context: Experiments were conducted in the presence of 1 mM selenite; despite optical density differences (attributed in part to elemental selenium formation), the authors report no differences in CFUs for at least 22 h between WT and mutants under selenite exposure. (avendano2023productionofselenium pages 5-6)

This provides recent, organism-specific evidence that sucA-linked central metabolism affects a real biotransformation phenotype (selenite → selenium nanoparticles). (avendano2023productionofselenium pages 5-6)

4.2 2024: multi-omics suggests metabolic rerouting around decarboxylative TCA steps

A 2024 Communications Biology multi-omics study of a defined synthetic co-culture (cyanobacterium feeding sucrose to P. putida) reports decreased protein abundances of SucA/B/D and Idh in P. putida alongside increased glyoxylate-cycle enzymes AceA and GlcB, interpreted as partial TCA “shutdown” and redirection through the glyoxylate cycle. (kratzl2024pseudomonasputidaas pages 7-9)

Quantitative transcriptomics in the same study reported idh transcript upregulation (log2-FC = 1.9) and aceA transcript downregulation (log2-FC = −2.64), emphasizing that transcript–protein concordance was only moderate and suggesting additional layers of regulation. (kratzl2024pseudomonasputidaas pages 7-9)

5) Current applications and real-world implementations

5.1 Biotechnology: nanoparticle biosynthesis

The 2023 KT2440 study indicates that manipulating central metabolic nodes including sucA can affect selenite biotransformation to selenium nanoparticles, and identifies gene targets of potential biotechnological interest for tuning nanoparticle formation rate. (avendano2023productionofselenium pages 5-6)

5.2 Systems and synthetic biology: managing redox and carbon flux

More generally, authoritative enzymology literature emphasizes OGDHc as a rate-limiting and regulatable node at the intersection of carbon flux and redox status. (bunik2013translatingenzymologyinto pages 2-3, bunik2013translatingenzymologyinto pages 1-2)

In applied contexts, the 2024 co-culture study illustrates a real implementation of multi-omics to diagnose central metabolism rewiring (including downshifts in SucA/B/D proteins) as part of designing or understanding microbial consortia. (kratzl2024pseudomonasputidaas pages 7-9)

6) Expert opinions and authoritative synthesis (interpretation anchored in reviews)

A detailed review of the OGDHc frames this complex as an irreversible, rate-limiting TCA step whose flux is tuned through enzyme–ligand interactions and protein–protein interactions, and notes that component stoichiometry and cofactor interactions can serve as regulatory handles. (bunik2013translatingenzymologyinto pages 1-2, bunik2013translatingenzymologyinto pages 2-3)

In the context of functional annotation for KT2440 sucA (Q88FA9), these authoritative sources justify annotating SucA as:
- ThDP-dependent decarboxylase E1 component of OGDHc
- critical for producing succinyl-CoA and NADH from 2-oxoglutarate
- likely a key control point linking carbon/nitrogen/redox metabolism

7) Statistics & data highlights (compiled)

• Proteomics (GraT toxin response, ΔgraA vs WT, 25 °C): SucA fold change −2.12; Idh −2.87; SucD −2.52. (ainelo2019pseudomonasputidaresponds media c323b5f8)
• Selenite conversion study: 1 mM selenite exposure; no CFU difference for ≥22 h between WT and mutants; sucA mutant shows slight reduction in growth/biomass. (avendano2023productionofselenium pages 5-6)
• Co-culture transcriptomics: idh transcript log2-FC = 1.9; aceA transcript log2-FC = −2.64; proteomics indicates decreased SucA/B/D proteins with increased glyoxylate-cycle enzymes. (kratzl2024pseudomonasputidaas pages 7-9)
• RNA–protein pulldown (sucA-II motif regulation hypothesis): condition-dependent changes in numbers of specifically bound proteins (e.g., 5 specific proteins in rich medium vs 17 in osmotic shock; 3 in rich vs 7 in nitrogen limitation), supporting stress-dependent post-transcriptional interaction landscapes. (geiger2019investigationofrnabased pages 65-68)

8) Limitations of the current evidence base (important for annotation quality)

• Direct biochemical characterization (e.g., KT2440 SucA kinetic parameters, in vitro substrate specificity across analogs) was not available in the retrieved KT2440 primary literature excerpts; catalytic mechanism is therefore best supported by conserved-family enzymology and cross-bacterial evidence. (chakraborty2022engineeringthe2oxoglutarate pages 2-3, bunik2013translatingenzymologyinto pages 2-3)
• RNA motif work proposes and tests regulatory hypotheses but does not conclusively establish a ligand-based riboswitch for KT2440 sucA regulation. (geiger2019investigationofrnabased pages 83-87, geiger2019investigationofrnabaseda pages 83-87)

Evidence summary table

Table: This table summarizes the main functional-annotation evidence for Pseudomonas putida KT2440 sucA (PP_4189; UniProt Q88FA9), covering enzymatic function, pathway placement, regulation, stress-linked phenotypes, and quantitative findings from the gathered literature.

References

1. (avendano2023productionofselenium pages 5-6): Roberto Avendaño, Said Muñoz‐Montero, Diego Rojas‐Gätjens, Paola Fuentes‐Schweizer, Sofía Vieto, Rafael Montenegro, Manuel Salvador, Rufus Frew, Juhyun Kim, Max Chavarría, and Jose I. Jiménez. Production of selenium nanoparticles occurs through an interconnected pathway of sulphur metabolism and oxidative stress response in pseudomonas putida kt2440. Microbial Biotechnology, 16:931-946, Jan 2023. URL: https://doi.org/10.1111/1751-7915.14215, doi:10.1111/1751-7915.14215. This article has 26 citations and is from a peer-reviewed journal.

2. (ainelo2019pseudomonasputidaresponds pages 5-9): Andres Ainelo, Rando Porosk, Kalle Kilk, Sirli Rosendahl, Jaanus Remme, and Rita Hõrak. Pseudomonas putida responds to the toxin grat by inducing ribosome biogenesis factors and repressing tca cycle enzymes. Toxins, 11:103, Feb 2019. URL: https://doi.org/10.3390/toxins11020103, doi:10.3390/toxins11020103. This article has 10 citations.

3. (bunik2013translatingenzymologyinto pages 2-3): Victoria I. Bunik, Guenter Raddatz, and Slawomir Strumilo. Translating enzymology into metabolic regulation: the case of the 2- oxoglutarate dehydrogenase multienzyme complex. Apr 2013. URL: https://doi.org/10.2174/2212796811307010008, doi:10.2174/2212796811307010008. This article has 24 citations and is from a peer-reviewed journal.

4. (chakraborty2022engineeringthe2oxoglutarate pages 2-3): Joydeep Chakraborty, Natalia Nemeria, Yujeong Shim, Xu Zhang, Elena L. Guevara, Hetal Patel, Edgardo T. Farinas, and Frank Jordan. Engineering the 2-oxoglutarate dehydrogenase complex to understand catalysis and alter substrate recognition. Reactions, 3:139-159, Feb 2022. URL: https://doi.org/10.3390/reactions3010011, doi:10.3390/reactions3010011. This article has 8 citations.

5. (bunik2013translatingenzymologyinto pages 1-2): Victoria I. Bunik, Guenter Raddatz, and Slawomir Strumilo. Translating enzymology into metabolic regulation: the case of the 2- oxoglutarate dehydrogenase multienzyme complex. Apr 2013. URL: https://doi.org/10.2174/2212796811307010008, doi:10.2174/2212796811307010008. This article has 24 citations and is from a peer-reviewed journal.

6. (bunik2013translatingenzymologyinto pages 3-4): Victoria I. Bunik, Guenter Raddatz, and Slawomir Strumilo. Translating enzymology into metabolic regulation: the case of the 2- oxoglutarate dehydrogenase multienzyme complex. Apr 2013. URL: https://doi.org/10.2174/2212796811307010008, doi:10.2174/2212796811307010008. This article has 24 citations and is from a peer-reviewed journal.

7. (bunik2013translatingenzymologyinto pages 4-6): Victoria I. Bunik, Guenter Raddatz, and Slawomir Strumilo. Translating enzymology into metabolic regulation: the case of the 2- oxoglutarate dehydrogenase multienzyme complex. Apr 2013. URL: https://doi.org/10.2174/2212796811307010008, doi:10.2174/2212796811307010008. This article has 24 citations and is from a peer-reviewed journal.

8. (chakraborty2022engineeringthe2oxoglutarate pages 1-2): Joydeep Chakraborty, Natalia Nemeria, Yujeong Shim, Xu Zhang, Elena L. Guevara, Hetal Patel, Edgardo T. Farinas, and Frank Jordan. Engineering the 2-oxoglutarate dehydrogenase complex to understand catalysis and alter substrate recognition. Reactions, 3:139-159, Feb 2022. URL: https://doi.org/10.3390/reactions3010011, doi:10.3390/reactions3010011. This article has 8 citations.

9. (geiger2019investigationofrnabased pages 18-22): S Geiger. Investigation of rna-based regulation of gene expression in proteobacterial energy metabolism. Unknown journal, 2019.

10. (kratzl2024pseudomonasputidaas pages 7-9): Franziska Kratzl, Marlene Urban, Jagroop Pandhal, Mengxun Shi, Chen Meng, Karin Kleigrewe, Andreas Kremling, and Katharina Pflüger-Grau. Pseudomonas putida as saviour for troubled synechococcus elongatus in a synthetic co-culture – interaction studies based on a multi-omics approach. Communications Biology, Apr 2024. URL: https://doi.org/10.1038/s42003-024-06098-5, doi:10.1038/s42003-024-06098-5. This article has 8 citations and is from a peer-reviewed journal.

11. (ainelo2019pseudomonasputidaresponds media c323b5f8): Andres Ainelo, Rando Porosk, Kalle Kilk, Sirli Rosendahl, Jaanus Remme, and Rita Hõrak. Pseudomonas putida responds to the toxin grat by inducing ribosome biogenesis factors and repressing tca cycle enzymes. Toxins, 11:103, Feb 2019. URL: https://doi.org/10.3390/toxins11020103, doi:10.3390/toxins11020103. This article has 10 citations.

12. (geiger2019investigationofrnabased pages 22-27): S Geiger. Investigation of rna-based regulation of gene expression in proteobacterial energy metabolism. Unknown journal, 2019.

13. (geiger2019investigationofrnabased pages 83-87): S Geiger. Investigation of rna-based regulation of gene expression in proteobacterial energy metabolism. Unknown journal, 2019.

14. (geiger2019investigationofrnabaseda pages 83-87): S Geiger. Investigation of rna-based regulation of gene expression in proteobacterial energy metabolism. Unknown journal, 2019.

15. (geiger2019investigationofrnabased pages 65-68): S Geiger. Investigation of rna-based regulation of gene expression in proteobacterial energy metabolism. Unknown journal, 2019.

16. (geiger2019investigationofrnabaseda pages 50-54): S Geiger. Investigation of rna-based regulation of gene expression in proteobacterial energy metabolism. Unknown journal, 2019.

17. (ainelo2019pseudomonasputidaresponds pages 1-3): Andres Ainelo, Rando Porosk, Kalle Kilk, Sirli Rosendahl, Jaanus Remme, and Rita Hõrak. Pseudomonas putida responds to the toxin grat by inducing ribosome biogenesis factors and repressing tca cycle enzymes. Toxins, 11:103, Feb 2019. URL: https://doi.org/10.3390/toxins11020103, doi:10.3390/toxins11020103. This article has 10 citations.

Artifacts

• Edison artifact artifact-00
  

Citations

1. bunik2013translatingenzymologyinto pages 1-2
2. bunik2013translatingenzymologyinto pages 2-3
3. geiger2019investigationofrnabased pages 18-22
4. ainelo2019pseudomonasputidaresponds pages 5-9
5. avendano2023productionofselenium pages 5-6
6. kratzl2024pseudomonasputidaas pages 7-9
7. geiger2019investigationofrnabased pages 65-68
8. bunik2013translatingenzymologyinto pages 3-4
9. bunik2013translatingenzymologyinto pages 4-6
10. geiger2019investigationofrnabased pages 22-27
11. geiger2019investigationofrnabased pages 83-87
12. geiger2019investigationofrnabaseda pages 83-87
13. geiger2019investigationofrnabaseda pages 50-54
14. ainelo2019pseudomonasputidaresponds pages 1-3
15. https://doi.org/10.1111/1751-7915.14215;
16. https://doi.org/10.3390/reactions3010011;
17. https://doi.org/10.2174/2212796811307010008
18. https://doi.org/10.3390/toxins11020103;
19. https://doi.org/10.1111/1751-7915.14215
20. https://doi.org/10.3390/toxins11020103
21. https://doi.org/10.1111/1751-7915.14215,
22. https://doi.org/10.3390/toxins11020103,
23. https://doi.org/10.2174/2212796811307010008,
24. https://doi.org/10.3390/reactions3010011,
25. https://doi.org/10.1038/s42003-024-06098-5,

Functional Annotation Report: sucA (Q88FA9 / PP_4189) in Pseudomonas putida KT2440

Gene: sucA · UniProt: Q88FA9 · Locus: PP_4189 · Organism: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / KT2440) · EC: 1.2.4.2

Summary

sucA (Q88FA9, ordered locus PP_4189) encodes the E1o component (2-oxoglutarate dehydrogenase, EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase complex (OGDHc / α-ketoglutarate dehydrogenase complex, KGDHc) in Pseudomonas putida KT2440. Its gene product is a thiamine-diphosphate (ThDP)-dependent oxidoreductase that catalyzes the first, committed and rate-limiting step of the fourth reaction of the tricarboxylic acid (TCA) cycle: the oxidative decarboxylation of 2-oxoglutarate (α-ketoglutarate). The enzyme decarboxylates its C5 keto-acid substrate to yield a covalent succinyl-ThDP enamine intermediate and CO₂, then transfers the succinyl moiety by reductive succinylation onto the lipoyl "swinging arm" of the E2 core (SucB). Together with E2 (dihydrolipoyl succinyltransferase, SucB) and E3 (dihydrolipoyl dehydrogenase, LpdG), the overall multienzyme complex converts 2-oxoglutarate + CoA + NAD⁺ → succinyl-CoA + CO₂ + NADH. This positions SucA at a pivotal carbon/nitrogen and redox/energy node, partitioning flux between respiratory ATP generation and biosynthetic precursor (glutamate/succinyl-CoA) supply.

The identity assignment is robust and passes all mandatory verification checks. The gene symbol sucA is the canonical bacterial designation for the E1o subunit; the UniProt description, EC number (1.2.4.2), protein family (alpha-ketoglutarate dehydrogenase), and InterPro domain architecture (2-oxogl_dehyd_N, 2oxoglutarate_DH_E1, DH_E1, KGD_C) all converge on this function. In P. putida KT2440's own genome, PP_4189 sits in a contiguous, co-oriented sdhA–sdhB–sucA–sucB–lpdG gene cluster that encodes all three OGDHc subunits (E1/E2/E3) side by side, exactly as expected for this operonic module of the TCA cycle.

Because no direct enzymology exists for the P. putida protein itself, its precise mechanism and substrate specificity are inferred with high confidence from its closest biochemically and structurally characterized ortholog, Escherichia coli SucA (65.6% amino-acid identity over the full-length single-chain α/β-fused polypeptide). The catalytically and regulatorily important residues — the active-site histidine cluster (E. coli His260/His298/His729 → P. putida His266/His304/His737) and the Thr405→Thr411 regulatory AMPylation site — are conserved, licensing functional transfer. The enzyme operates in the bacterial cytoplasm as a soluble, high-molecular-weight multienzyme assembly.

Gene/Protein Identity Verification

Verified — identity is unambiguous. All four mandatory checks pass:

1. Symbol matches function. sucA is the canonical bacterial gene name for the E1o (2-oxoglutarate dehydrogenase) subunit; this matches the UniProt RecName "2-oxoglutarate dehydrogenase E1 component."
2. Organism confirmed. The target is Pseudomonas putida KT2440, and PP_4189 is confirmed as sucA within KT2440's own genome (KEGG organism ppu).
3. Family/domains align. The alpha-ketoglutarate dehydrogenase family and InterPro domains (2oxoglutarate_DH_E1 / DH_E1 / KGD_C) match the literature on E1o enzymes.
4. No same-symbol confusion. The well-characterized E. coli SucA used for functional transfer is a genuine 65.6%-identical ortholog — not an unrelated gene sharing the symbol.

Key Findings

F001 — SucA is the ThDP-dependent E1o decarboxylase (EC 1.2.4.2)

UniProt Q88FA9 is annotated as RecName: 2-oxoglutarate dehydrogenase E1 component, EC 1.2.4.2, in the alpha-ketoglutarate dehydrogenase family, with defining InterPro domains IPR011603 (2oxoglutarate_DH_E1), IPR001017 (DH_E1), and IPR031717 (KGD C-terminal). The gene symbol sucA is the canonical bacterial name for this E1 subunit. The bacterial E1o homolog is a thiamine-diphosphate–dependent enzyme that catalyzes the first, rate-limiting step of the OGDH complex reaction.

This is directly supported by the E. coli structural work of Frank et al.: "The thiamine-dependent E1o component (EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase complex catalyses a rate-limiting step of the tricarboxylic acid cycle (TCA) of aerobically respiring organisms" (PMID: 17367808). This single sentence anchors the enzyme identity, the cofactor (thiamine diphosphate), the EC number (1.2.4.2), and the physiological role as the rate-limiting E1 step that sucA performs.

F002 — SucA acts within a multienzyme OGDH complex using lipoyl-arm substrate channeling

SucA does not act in isolation. It is the E1 catalytic subunit of the OGDH/KGDH complex, which consists of multiple copies of three catalytic subunits: E1 (SucA, 2-oxoglutarate dehydrogenase, EC 1.2.4.2), E2 (SucB, dihydrolipoyl succinyltransferase, EC 2.3.1.61) forming the structural core, and E3 (LpdG, dihydrolipoamide dehydrogenase, EC 1.8.1.4). E1 decarboxylates the substrate and passes the succinyl group to lipoyl moieties tethered on the E2 core via a flexible "swinging arm."

Kinetic and computer-model analysis of the E. coli complex demonstrates that this channeling is highly interconnected: "More than one lipoyl moiety services each E1 subunit (alpha-ketoglutarate dehydrogenase, EC 1.2.4.2), and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present" (PMID: 6403946). The three-subunit architecture is confirmed independently: "The complex consists of multiple copies of three catalytic subunits: α-ketoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2) and dihydrolipoamide dehydrogenase (E3)" (PMID: 36256893). SucA is thus the point-of-entry catalyst in a nanomachine that couples decarboxylation, acyl transfer, and NADH generation through substrate channeling.

F003 — Structure defines substrate specificity (His/Ser cluster) and allosteric control (oxaloacetate, AMP)

The 2.6 Å crystal structure of E. coli E1o reveals the ThDP-binding fold and an α₂β₂-like architecture in which the α and β subunits are fused into a single polypeptide — consistent with Q88FA9 being a single ~933–943-residue chain rather than two separate subunits. As Frank et al. state, "the alpha and beta subunits are fused as a single polypeptide" (PMID: 17367808).

The same structure identifies the determinants of substrate specificity and regulation: "The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate. Oxaloacetate inhibits E1o activity at physiological concentrations" (PMID: 17367808). Additionally, an AMP molecule is specifically recognized in a pocket linking the enzyme's domains, providing a second layer of allosteric/energy-state control. These features are the molecular basis both for 2-oxoglutarate selectivity and for feedback sensing of TCA metabolite pools.

F004 — Genomic and transcriptional coupling to the succinate-branch TCA genes

In E. coli, sucA lies in the suc operon together with the E2o (sucB) and succinyl-CoA synthetase (sucCD) genes, within the larger gltA–sdhCDAB–sucABCD citric-acid-cycle gene cluster. As reported: "the suc operon, which also encodes the dehydrogenase (Elo; sucA) and succinyltransferase (E2o; sucB) components of the 2-oxoglutarate dehydrogenase complex" (PMID: 3543212). The sucABCD genes are coordinately regulated, being controlled largely from the upstream sdhC promoter with negative control under anaerobiosis by ArcA and Fnr: "The sucABCD genes of Escherichia coli encode subunits for two enzymes of the tricarboxylic acid (TCA) cycle, alpha-ketoglutarate dehydrogenase (sucAB) and succinyl coenzyme A synthetase (sucCD)" (PMID: 9209026).

The functional consequence of losing this activity is demonstrable: "disruption of the sucA or sucB gene (encoding subunits of the 2-oxoglutarate dehydrogenase complex) notably decreased HHP survival" (PMID: 30497599), establishing that SucA's OGDHc function has a measurable physiological phenotype. In P. putida KT2440, the citric acid cycle is a component of tightly regulated central carbon metabolism, and sucA corresponds to locus PP_4189.

F005 — P. putida SucA is a close ortholog of biochemically characterized E. coli SucA (65.6% identity)

A pairwise global alignment (Needleman-Wunsch, +2/−1/−1) of the full-length UniProt sequences shows P. putida KT2440 SucA (Q88FA9, 943 aa) and E. coli K-12 SucA (P0AFG3 / ODO1_ECOLI, 933 aa) share 588 identical residues over 896 aligned columns = 65.6% amino-acid identity. Both are annotated "2-oxoglutarate dehydrogenase E1 component," and both are single long polypeptides with α-like and β-like domains fused — matching the E1o architecture solved crystallographically in E. coli: "the alpha and beta subunits are fused as a single polypeptide" (PMID: 17367808). This high sequence identity across the full length, spanning the ThDP-binding and catalytic regions, provides a strong, rigorous justification for transferring the E. coli mechanism, substrate specificity, and structure to the P. putida ortholog.

F006 — Substrate specificity for 2-oxoglutarate (C5), distinct from the 2-oxoadipate E1a paralog

The enzyme is specifically dedicated to 2-oxoglutarate, the C5 substrate of the TCA cycle's fourth step, and is functionally distinct from the paralogous 2-oxoadipate dehydrogenase E1a (DHTKD1). The reaction "catalyzes the fourth step of the tricarboxylic acid (TCA) cycle and links carbohydrate, fatty acid and amino acid metabolism to the aerobic production of ATP" (PMID: 41722662), converting α-ketoglutarate to succinyl-CoA with NADH generation.

Substrate specificity within the E1 family is enzyme-specific and can be quantified: the human 2-oxoadipate dehydrogenase E1a shows "an approximately 49-fold preference in catalytic efficiency for OA over OG, indicating that hE1a is specific to the 2-oxoadipate dehydrogenase complex" (PMID: 29191460). By contrast, the canonical E1o (sucA-type) is the 2-oxoglutarate-specific component that recruits the shared E2o/E3 machinery. This ~49-fold catalytic-efficiency discrimination in the paralog underscores that E1 substrate selection is precise, and confirms SucA's dedicated role as the 2-oxoglutarate (not 2-oxoadipate) enzyme.

F007 — In P. putida KT2440, PP_4189 sits in a contiguous sdhAB–sucA–sucB–lpdG cluster

KEGG genome annotation (organism ppu) places five co-oriented genes contiguously on the complement strand of the P. putida KT2440 chromosome:

Intergenic gaps are small (~40–95 bp), consistent with an operon. KEGG assigns PP_4189 to Citrate cycle (ppu00020), Lipoic acid metabolism (ppu00785), 2-Oxocarboxylic acid metabolism (ppu01210), and Carbon metabolism (ppu01200), and to modules M00009 (TCA cycle) and M00011 (2-oxoglutarate ⇒ oxaloacetate, second oxidation). This confirms — in P. putida's own genome, not merely by analogy — that all three OGDHc catalytic subunits (E1/E2/E3) are encoded side by side, physically clustering the machinery SucA works within.

F008 — Catalytic and regulatory residues are conserved in P. putida SucA

Alignment mapping of E. coli SucA (P0AFG3) onto P. putida SucA (Q88FA9) shows uniformly high domain-wise identity — the N-terminal region (res 1–350) is 64.0% identical and the central+C-terminal catalytic region (351–933) is 66.5% identical — with 71% of all E. coli histidines conserved as His. Crucially, the histidines corresponding to the E. coli E1o active-site region are conserved: E. coli His260/His298/His729 → P. putida His266/His304/His737. This maps the specificity-conferring active-site cluster identified crystallographically — "The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate" (PMID: 17367808) — directly onto the P. putida sequence.

In addition, the UniProt-annotated regulatory modification site of E. coli SucA, Thr405 (O-AMP-threonine, AMPylated by the YdiU/SelO protein AMPylase), aligns to a conserved threonine (Thr411) in P. putida SucA. This indicates conservation not only of catalysis but also of a post-translational regulatory mechanism, suggesting SucA activity in P. putida may likewise be modulated by AMPylation under stress conditions.

Mechanistic Model / Interpretation

The reaction and its place in the cycle

SucA catalyzes the entry step of the OGDHc-catalyzed overall reaction:

  2-oxoglutarate + CoA-SH + NAD+  ──►  succinyl-CoA + CO2 + NADH + H+
  (α-ketoglutarate, C5)                (C4-acyl-CoA)

The complex partitions this into three sequential, channeled half-reactions across the three subunits:

  ┌────────────────────────── OGDH COMPLEX (soluble, cytoplasm) ──────────────────────────┐
  │                                                                                        │
  │  E1o = SucA (PP_4189)          E2o = SucB (PP_4188)          E3 = LpdG (PP_4187)        │
  │  ThDP-dependent                lipoyl-bearing core           FAD + NAD+                 │
  │  ───────────────               ──────────────────           ──────────────             │
  │                                                                                        │
  │  2-oxoglutarate                                                                        │
  │       │ (1) decarboxylation                                                            │
  │       ▼   ThDP → succinyl-ThDP  + CO2                                                   │
  │  [succinyl-ThDP enamine]                                                                │
  │       │ (2) reductive succinylation                                                    │
  │       └──────────────►  lipoyl-S-succinyl (E2)                                          │
  │                              │ (3) transfer to CoA                                      │
  │                              └──────────►  succinyl-CoA                                 │
  │                         dihydrolipoyl (E2)                                              │
  │                              │ (4) reoxidation                                          │
  │                              └──────────►  E3: FADH2 → NADH                             │
  │                                                                                        │
  └────────────────────────────────────────────────────────────────────────────────────┘
                   Step 4 of the TCA cycle · rate-limiting · irreversible

Step 1 (SucA/E1o): ThDP attacks the C2 keto-carbon of 2-oxoglutarate; decarboxylation releases CO₂ and generates a resonance-stabilized succinyl-ThDP enamine (carbanion) intermediate. The three-histidine + serine active-site cluster (P. putida His266/His304/His737) orients and stabilizes the C5 dicarboxylate substrate — the structural basis for selecting 2-oxoglutarate over other 2-oxo acids such as 2-oxoadipate (the DHTKD1/E1a substrate). This is the committed, rate-limiting, essentially irreversible step.

Step 2 (SucA→SucB): The succinyl group is transferred by reductive succinylation onto the oxidized lipoamide arm of E2, reducing the disulfide. The flexible lipoyl "swinging arm" then visits the E2 acyltransferase site.

Step 3 (SucB/E2o): The succinyl group is transferred from dihydrolipoamide to CoA, releasing succinyl-CoA — a high-energy thioester used downstream by succinyl-CoA synthetase for substrate-level phosphorylation.

Step 4 (LpdG/E3): The reduced dihydrolipoamide is reoxidized by E3 (FAD), regenerating oxidized lipoamide and reducing NAD⁺ to NADH, which feeds oxidative phosphorylation.

Regulation and metabolic significance

SucA sits at a triple control point:

1. Rate-limiting kinetics — the E1o step gates overall flux through the second half of the TCA cycle.
2. Allosteric feedback — the active site can accommodate and be inhibited by oxaloacetate at physiological concentrations, and an AMP-binding pocket senses energy charge, coupling activity to the cell's metabolic state (from F003).
3. Post-translational modification — the conserved Thr411 AMPylation site (F008) implies regulation by the YdiU/SelO AMPylase, a stress-responsive mechanism.

Metabolically, 2-oxoglutarate is the principal carbon/nitrogen crossroads: it is the acceptor of ammonia in glutamate/glutamine synthesis and the exit point for amino-acid-derived carbon into the TCA cycle. By controlling the drain of 2-oxoglutarate toward succinyl-CoA, SucA partitions flux between (a) respiratory energy generation (NADH → oxidative phosphorylation; succinyl-CoA → substrate-level phosphorylation) and (b) biosynthetic precursor supply (retaining 2-oxoglutarate for nitrogen assimilation and glutamate-family amino acids). In P. putida KT2440, whose central carbon metabolism is noted for tight, largely post-transcriptional/metabolic regulation (PMID: 24951791), this node is especially important for balancing the organism's versatile catabolic lifestyle with biosynthesis.

Localization

The OGDHc, including SucA, is a soluble cytoplasmic multienzyme complex in bacteria (the analog of the mitochondrial-matrix complex in eukaryotes). There are no signal peptides, transmembrane segments, or secretion signals in the annotation; the enzyme carries out its function in the cytosol, where it associates non-covalently around the E2 core.

Evidence Base

Additional reviewed literature (PMIDs 28579060, 27986918, 20228444, 11368334, 6341609, 25184115, 40609475, 39299525, 1730230) provides supporting context on E3 deficiency, complex reengineering, random steady-state channeling models, the E1 ping-pong mechanism, KGDC quaternary heterogeneity, and ROS generation — reinforcing the general mechanistic model of 2-oxo-acid dehydrogenase complexes within which SucA functions.

Direct experimental evidence exists for the E. coli ortholog (crystal structure, kinetics, operon regulation, knockout phenotypes). Inference from evolution/structure (65.6% identity, conserved active-site and regulatory residues) transfers this to P. putida. Bioinformatic/genomic evidence (KEGG operon annotation) confirms the complete complex is encoded in P. putida itself.

Limitations and Knowledge Gaps

1. No direct enzymology of PP_4189. There is no published biochemical characterization (purified enzyme, kinetics, K_m, k_cat, crystal structure) of the P. putida KT2440 SucA protein specifically. All mechanistic and substrate-specificity claims rest on orthology transfer from E. coli (65.6% identity) and the broader alpha-ketoglutarate dehydrogenase family.

2. Substrate specificity is inferred, not measured, for the target. While the active-site His cluster is conserved, the possibility of a broadened or altered substrate range (e.g., partial activity toward other 2-oxo acids) has not been experimentally excluded in P. putida.

3. Regulation is largely extrapolated. Oxaloacetate inhibition, AMP binding, and Thr411 AMPylation are established or predicted from E. coli/UniProt annotation. Their operation and physiological relevance in P. putida KT2440 (which relies heavily on post-transcriptional/metabolic regulation, PMID: 24951791) remain to be tested.

4. Complex stoichiometry and quaternary structure in P. putida are unknown; the E. coli complex itself may be structurally heterogeneous (PMID: 6341609), so precise E1:E2:E3 ratios in P. putida are unconfirmed.

5. Operon transcription is inferred from gene contiguity and small intergenic gaps; the actual transcriptional unit boundaries, promoters, and regulators for the P. putida sdhAB–sucAB–lpdG cluster have not been experimentally mapped.

Proposed Follow-up Experiments / Actions

1. Recombinant expression and kinetic characterization. Clone PP_4189 (± PP_4188/PP_4187), purify, and reconstitute the OGDHc in vitro. Measure K_m and k_cat for 2-oxoglutarate and, as controls, 2-oxoadipate and pyruvate to directly confirm substrate specificity and quantify discrimination — mirroring the DHTKD1/E1a comparison (PMID: 29191460).

2. Structural determination. Solve the crystal or cryo-EM structure of P. putida SucA (or the whole complex) to verify the predicted single-chain α/β fold and the His266/His304/His737 active-site geometry, and to test oxaloacetate/AMP binding pockets predicted from the E. coli structure (PMID: 17367808).

3. Gene knockout / conditional depletion. Construct a PP_4189 deletion or knockdown in KT2440 and characterize growth on TCA-cycle-dependent carbon sources, glutamate/nitrogen handling, redox balance, and stress survival (e.g., analogous to the HHP phenotype in E. coli, PMID: 30497599).

4. Test AMPylation regulation. Verify whether Thr411 is AMPylated in vivo by the P. putida YdiU/SelO homolog and whether this modulates activity under nutrient/stress transitions — a targeted test of the conserved regulatory site (F008).

5. Operon mapping. Use RT-PCR/RNA-seq and promoter-reporter fusions to define the transcriptional unit(s) spanning sdhAB–sucAB–lpdG and identify regulators (e.g., anaerobiosis/oxygen control analogous to ArcA/Fnr in E. coli, PMID: 9209026).

6. Metabolic flux analysis. Use ¹³C-labeling in KT2440 to quantify flux partitioning at the 2-oxoglutarate node between respiratory oxidation (via SucA) and nitrogen assimilation/biosynthesis, directly probing SucA's role as the carbon/nitrogen branch-point gate.

Artifacts

• OpenScientist final report
• OpenScientist final report

Citations

1. PMID:17367808
2. PMID:6403946
3. PMID:36256893
4. PMID:3543212
5. PMID:9209026
6. PMID:30497599
7. PMID:41722662
8. PMID:29191460
9. PMID:24951791
10. PMID:6341609