Beta-ketoadipyl-CoA thiolase of the protocatechuate branch of the beta-ketoadipate pathway. PcaF-I catalyzes thiolytic cleavage of 3-oxoadipyl-CoA to succinyl-CoA and acetyl-CoA, completing assimilation of 3,4-dihydroxybenzoate-derived carbon into central metabolism. Available evidence supports a degradative thiolase specialized for aromatic compound catabolism rather than a general lipid-metabolism enzyme.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0003988
acetyl-CoA C-acyltransferase activity
|
IEA
GO_REF:0000117 |
KEEP AS NON CORE |
Summary: Correct as a broad parent activity for a thiolase, but it is less informative than the specific 3-oxoadipyl-CoA thiolase term already present.
Reason: PcaF-I is a C-acyltransferase/thiolase, but the curated core function should use GO:0033812 to capture substrate specificity.
|
|
GO:0016746
acyltransferase activity
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: Too general to be useful for this gene and likely inherited from family-level domain mappings rather than pathway-specific evidence.
Reason: The specific thiolase activity is already represented by GO:0033812, so this broad umbrella term adds little biological information.
|
|
GO:0016747
acyltransferase activity, transferring groups other than amino-acyl groups
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: Mechanistically true at a very high level, but still a redundant broad family term relative to the specific 3-oxoadipyl-CoA thiolase annotation.
Reason: Pathway-specific curation should prefer the substrate-resolved thiolase term.
|
|
GO:0033812
3-oxoadipyl-CoA thiolase activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: Best current molecular function term for PcaF-I and consistent with UniProt pathway assignment, classical pcaF genetics, KT2440 proteomics, and the KT2440 structural degradative-thiolase paper.
Reason: This term captures the specific beta-ketoadipyl-CoA/3-oxoadipyl-CoA thiolase activity that defines the enzyme.
|
|
GO:0006629
lipid metabolic process
|
IEA
GO_REF:0000117 |
REMOVE |
Summary: Not supported as a core role for this gene. The enzyme functions in aromatic compound degradation through the beta-ketoadipate pathway, not in canonical lipid metabolism.
Reason: This appears to be a family-level over-projection from thiolase chemistry rather than a pathway-specific assignment for PcaF-I.
|
|
GO:0019619
3,4-dihydroxybenzoate catabolic process
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: Appropriate biological-process annotation because PcaF-I acts in the shared beta-ketoadipate segment used to complete protocatechuate/3,4-dihydroxybenzoate degradation.
Reason: Proteomics and classical pca pathway genetics support participation of PcaF in the protocatechuate branch of aromatic compound catabolism.
|
Q: Is PcaF-I dedicated to beta-ketoadipate turnover in vivo, or can it efficiently process alternative CoA thioesters under physiological conditions?
Q: How is pcaF-I coordinated transcriptionally with the rest of the protocatechuate branch in KT2440 under mixed aromatic substrates?
Experiment: Construct a clean pcaF-I deletion in KT2440 and test growth plus metabolite accumulation on protocatechuate, p-hydroxybenzoate, vanillate, and benzoate to confirm the pathway block at the 3-oxoadipyl-CoA cleavage step.
Hypothesis: Loss of pcaF-I will prevent efficient flux through the beta-ketoadipate pathway and cause accumulation of upstream protocatechuate-branch intermediates.
Experiment: Purify PcaF-I and compare catalytic efficiency for 3-oxoadipyl-CoA versus alternative acyl-CoA substrates to quantify whether the enzyme is pathway-specialized or broadly permissive like other degradative thiolases.
Hypothesis: PcaF-I will show clear catalytic preference for 3-oxoadipyl-CoA despite sharing general thiolase-family chemistry with broader acyltransferases.
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.
The UniProt accession Q88N39 corresponds to Pseudomonas putida KT2440 locus PP_1377 annotated as PcaF (PcaF-I), a β-ketoadipyl-CoA thiolase (syn. 3‑oxoadipyl‑CoA thiolase; EC 2.3.1.174). This identity is consistent with experimental KT2440 literature describing pcaF as encoding the thiolase catalyzing the final cleavage of β‑ketoadipyl‑CoA to acetyl‑CoA and succinyl‑CoA, and with functional complementation of a KT2440 ΔpcaF mutant by a homologous thiolase (paaE) (https://doi.org/10.1099/mic.0.2006/002444-0; published Feb 2007) (nogales2007characterizationofthe pages 6-7, nogales2007characterizationofthe pages 4-5).
The β‑ketoadipate pathway is a convergent aerobic catabolic route that funnels diverse aromatic compounds (including lignin-related aromatics and benzoate/protocatechuate intermediates) into a small set of ring‑cleavage products that are converted to β‑ketoadipate and then into central metabolites that enter the TCA cycle. In P. putida, the pathway is highlighted as a native, atom‑efficient route that can (under engineered conditions) convert lignin‑related aromatics to β‑ketoadipate at 1 mol product per mol aromatic substrate (https://doi.org/10.1126/sciadv.adj0053; published Sep 2023) (werner2023ligninconversionto pages 1-2).
PcaF is the β‑ketoadipyl‑CoA (3‑oxoadipyl‑CoA) thiolase catalyzing thiolytic cleavage of β‑ketoadipyl‑CoA to succinyl‑CoA and acetyl‑CoA (EC 2.3.1.174). In KT2440, this reaction is experimentally supported by product identification (HPLC) and spectrophotometric monitoring of the β‑ketoadipyl‑CoA–Mg2+ complex in crude extract assays (https://doi.org/10.1099/mic.0.2006/002444-0; Feb 2007) (nogales2007characterizationofthe pages 4-5).
Nogales et al. characterized the terminal thiolase activity in KT2440 using β‑ketoadipyl‑CoA generated by PcaIJ (β‑ketoadipyl‑CoA transferase) and showed that thiolase activity yields acetyl‑CoA and succinyl‑CoA, detected by HPLC with a decrease in β‑ketoadipyl‑CoA signal (https://doi.org/10.1099/mic.0.2006/002444-0; Feb 2007) (nogales2007characterizationofthe pages 4-5, nogales2007characterizationofthe pages 6-7).
In crude extracts, the KT2440 PcaF activity reported under the assay conditions was ~0.06 U per mg protein from cells grown on 4‑hydroxybenzoate (https://doi.org/10.1099/mic.0.2006/002444-0; Feb 2007) (nogales2007characterizationofthe pages 6-7). This paper did not report purified-enzyme kinetic constants (e.g., Km, kcat) for KT2440 PcaF in the retrieved excerpts, so current quantitative understanding for substrate specificity/kinetics remains primarily at the level of crude-extract activity and pathway-level function rather than detailed enzymology in this evidence set (nogales2007characterizationofthe pages 4-5).
Nogales et al. constructed a KT2440 ΔpcaF strain (KT2440dpcaF) and used it to (i) generate β‑ketoadipyl‑CoA via the intact PcaIJ transferase and (ii) confirm that loss of pcaF removes the thiolase step while leaving transferase activity present (https://doi.org/10.1099/mic.0.2006/002444-0; Feb 2007) (nogales2007characterizationofthe pages 4-5). They further showed that a homologous thiolase gene (paaE) shares high similarity to KT2440 PcaF and complements the KT2440 ΔpcaF phenotype, providing strong functional equivalence evidence for the thiolase reaction assignment (https://doi.org/10.1099/mic.0.2006/002444-0; Feb 2007) (nogales2007characterizationofthe pages 6-7, nogales2007characterizationofthe pages 2-4).
No direct subcellular localization experiment for KT2440 PcaF (e.g., fractionation, microscopy tagging) was found in the retrieved sources. However, PcaF activity was measured in crude cell extracts (soluble lysates) from 4‑hydroxybenzoate-grown KT2440 cultures, consistent with a cytosolic enzyme acting on CoA thioesters in intracellular metabolism (https://doi.org/10.1099/mic.0.2006/002444-0; Feb 2007) (nogales2007characterizationofthe pages 4-5).
In the canonical β‑ketoadipate branch, β‑ketoadipate is activated to a CoA thioester by CoA‑transferase activity (often pcaI/pcaJ in pseudomonads), and the resulting β‑ketoadipyl‑CoA is then cleaved by PcaF to acetyl‑CoA + succinyl‑CoA (nogales2007characterizationofthe pages 4-5). This endpoint is also referenced in systems-level analyses of aromatic catabolism: Borchert et al. interpret fitness modules for benzoate catabolism as requiring assimilation of acetyl‑CoA “produced by PcaF,” linking PcaF’s biochemical output to central metabolic adaptations such as the glyoxylate shunt (https://doi.org/10.1128/msystems.00942-23; published Mar 2024) (borchert2024machinelearninganalysis pages 6-7).
Direct KT2440-specific operon mapping for PP_1377 (pcaF-I) was not found in the retrieved evidence set. Comparative evidence from a related Pseudomonas aromatic-catabolic cluster shows the thiolase gene (catF; a pcaF ortholog) adjacent to and even overlapping a CoA-transferase gene (catJ) by 4 bp, consistent with tight clustering of transferase/thiolase steps in some pseudomonads (https://doi.org/10.1128/jb.184.1.216-223.2002; published Jan 2002) (gobel2002degradationofaromatics pages 1-2). This supports the plausibility of physical clustering with CoA-transferase components but should not be treated as a definitive KT2440 genomic arrangement without KT2440-specific mapping evidence.
A major recent development is the engineering of P. putida KT2440 to funnel lignin-related aromatics (e.g., p-coumarate and ferulate) to β‑ketoadipate at industrially relevant metrics. Werner et al. engineered KT2440 to accumulate β‑ketoadipate by deleting pcaIJ (blocking downstream consumption), tuned upstream steps (O‑demethylation, hydroxylation, ring-opening), and deleted the global regulator crc to improve substrate utilization (https://doi.org/10.1126/sciadv.adj0053; Sep 2023) (werner2023ligninconversionto pages 2-4, werner2023ligninconversionto pages 1-2).
Key statistics from this 2023 work include:
- ~44.5 g/L β‑ketoadipate titer (model lignin-related compounds) and ~25 g/L from corn stover-derived lignin-related compounds (Sep 2023) (werner2023ligninconversionto pages 2-4, werner2023ligninconversionto pages 1-2).
- Productivities reported include 1.15 g/L/h (model LRCs) and 0.66 g/L/h (corn stover-derived), and an overall yield of 0.10 g product per g lignin in the lignin-derived stream (werner2023ligninconversionto pages 2-4, werner2023ligninconversionto pages 1-2).
- In some bioreactor comparisons within the same study: DO-stat achieved 43.5 ± 1.6 g/L at 0.55 ± 0.02 g/L/h, while constant fed-batch achieved 44.5 ± 1.85 g/L at 0.85 ± 0.04 g/L/h (werner2023ligninconversionto pages 2-4).
Although PcaF is downstream of the engineered production node (the strains were designed to block β‑ketoadipate consumption at pcaIJ), these results reinforce the centrality of the β‑ketoadipate pathway architecture in KT2440 and the importance of understanding downstream steps—including the PcaF thiolase endpoint—when engineering strains either for complete mineralization (native role) or for product accumulation (engineered role) (werner2023ligninconversionto pages 1-2).
The pathway rationale and deletion point are shown in the pathway schematic in Werner et al. (Figure showing the pcaIJ block for β‑ketoadipate accumulation) (werner2023ligninconversionto media ac5a69ec), and the reported titers/productivity trajectories are presented in their bioreactor performance figures (werner2023ligninconversionto media 88d7ff26).
Borchert et al. (Mar 2024) developed a machine learning approach (independent component analysis) over RB‑TnSeq fitness data in KT2440 and recovered functional modules including aromatic catabolism, explicitly connecting benzoate catabolism physiology to acetyl‑CoA assimilation demands that they attribute to PcaF output (https://doi.org/10.1128/msystems.00942-23; Mar 2024) (borchert2024machinelearninganalysis pages 6-7). This reflects a trend toward integrating large-scale fitness compendia with metabolic interpretation to accelerate annotation and guide engineering.
The clearest KT2440 real-world implementation tied to the β‑ketoadipate pathway is production of β‑ketoadipate (a polymer precursor) from lignin-derived aromatics via metabolic engineering and bioprocess development. The study reports technoeconomic analysis suggesting economic relevance, and demonstrates performance metrics (titer, productivity, yield) at scales/conditions characteristic of industrial bioreactor development rather than small-scale shake-flask proof-of-concept (https://doi.org/10.1126/sciadv.adj0053; Sep 2023) (werner2023ligninconversionto pages 2-4, werner2023ligninconversionto pages 8-10).
From an expert metabolic-systems perspective, PcaF’s key functional significance is that it converts the aromatic-funneling endpoint (β‑ketoadipyl‑CoA) into two central-CoA metabolites (acetyl‑CoA and succinyl‑CoA). Systems-level analyses interpret downstream central metabolism (e.g., glyoxylate shunt induction) as being shaped by the need to assimilate acetyl‑CoA generated at this step during growth on aromatics such as benzoate (https://doi.org/10.1128/msystems.00942-23; Mar 2024) (borchert2024machinelearninganalysis pages 6-7). Conversely, product-accumulation engineering often blocks earlier (e.g., pcaIJ deletion) to prevent flux into the PcaF step and the TCA cycle, enabling extracellular accumulation of β‑ketoadipate at high titer (https://doi.org/10.1126/sciadv.adj0053; Sep 2023) (werner2023ligninconversionto pages 1-2, werner2023ligninconversionto media ac5a69ec).
| Gene/protein | Reaction / function | Pathway role in P. putida KT2440 | Experimental evidence type | Key quantitative data | Source (year, DOI URL, citation id) |
|---|---|---|---|---|---|
| PcaF (UniProt Q88N39; PP_1377; pcaF-I) | β-ketoadipyl-CoA thiolase / 3-oxoadipyl-CoA thiolase; catalyzes thiolytic cleavage of β-ketoadipyl-CoA (3-oxoadipyl-CoA) to succinyl-CoA + acetyl-CoA | Terminal thiolase step of the β-ketoadipate branch of aromatic catabolism; links ring-cleavage products to central metabolism | Enzymatic assay in crude extracts; HPLC product identification; targeted mutant construction | PcaF activity in KT2440 crude extracts grown on 4-hydroxybenzoate: ~0.06 U mg⁻¹ protein; assays at 30 °C, pH 8.0; β-ketoadipyl-CoA HPLC retention time ~8.9 min | Nogales et al., 2007, https://doi.org/10.1099/mic.0.2006/002444-0 (nogales2007characterizationofthe pages 4-5) |
| PcaF (KT2440 ΔpcaF background) | Same reaction; loss of native thiolase function used to test functional equivalence of homologs | Confirms that pcaF is required for the final β-ketoadipyl-CoA cleavage step in β-ketoadipate-related metabolism | Directed insertional disruption of pcaF in KT2440; complementation with paaE homolog from E. coli W | Homologous PaaE shares 71% identity with PcaF and complements KT2440 ΔpcaF; homolog thiolase specific activity ~0.11 U mg⁻¹ in E. coli W grown on phenylacetate | Nogales et al., 2007, https://doi.org/10.1099/mic.0.2006/002444-0 (nogales2007characterizationofthe pages 6-7, nogales2007characterizationofthe pages 2-4) |
| PcaF | Produces acetyl-CoA and succinyl-CoA from β-ketoadipyl-CoA during benzoate/protocatechuate utilization | Downstream of protocatechuate catabolism; acetyl-CoA output is linked to induction of glyoxylate shunt functions during benzoate growth | RB-TnSeq fitness compendium analyzed by independent component analysis (functional module inference) | No direct enzyme kinetics reported; systems-level evidence connects PcaF-derived acetyl-CoA with benzoate-catabolic module behavior across 179 conditions | Borchert et al., 2024, https://doi.org/10.1128/msystems.00942-23 (borchert2024machinelearninganalysis pages 6-7) |
| β-ketoadipate pathway context (downstream of PcaF) | Native route funnels lignin-related aromatics to β-ketoadipate, which is normally further catabolized to TCA-cycle precursors | PcaF acts after β-ketoadipate CoA-transferase (PcaIJ); engineering commonly blocks at pcaIJ, not pcaF, to accumulate β-ketoadipate upstream of the thiolase step | Metabolic engineering + fed-batch bioprocess development in KT2440 | Engineered strains blocking downstream metabolism at pcaIJ reached 44.5 g L⁻¹ β-ketoadipate and 1.15 g L⁻¹ h⁻¹ from model lignin-related compounds; 25 g L⁻¹ and 0.66 g L⁻¹ h⁻¹ from corn stover-derived streams | Werner et al., 2023, https://doi.org/10.1126/sciadv.adj0053 (werner2023ligninconversionto pages 2-4, werner2023ligninconversionto pages 1-2, werner2023ligninconversionto media ac5a69ec) |
| β-ketoadipate pathway context (process interpretation) | High-flux aromatic funneling demonstrates the practical importance of the native Pca pathway that ultimately feeds acetyl-CoA/succinyl-CoA generation | Shows why the PcaF-catalyzed endpoint is central for lignin valorization, even when product-accumulation strains interrupt the pathway one step earlier | Bioreactor performance analysis; pathway schematic and productivity curves | DO-stat: 43.5 ± 1.6 g L⁻¹ β-ketoadipate at 0.55 ± 0.02 g L⁻¹ h⁻¹; constant fed-batch: 44.5 ± 1.85 g L⁻¹ at 0.85 ± 0.04 g L⁻¹ h⁻¹ in one comparison; article also reports up to 1.15 g L⁻¹ h⁻¹ and 0.10 g product g⁻¹ lignin overall yield | Werner et al., 2023, https://doi.org/10.1126/sciadv.adj0053 (werner2023ligninconversionto pages 2-4, werner2023ligninconversionto pages 8-10, werner2023ligninconversionto pages 1-2, werner2023ligninconversionto media 88d7ff26) |
| PcaF orthologous context in pseudomonads | Conserved 3-oxoadipyl-CoA thiolase identity in pca/cat gene clusters supports annotation of KT2440 PP_1377 as a thiolase-family β-ketoadipate enzyme | Comparative support for gene-family assignment and expected substrate specificity | Gene cloning / comparative sequence analysis in Pseudomonas sp. B13 | No KT2440-specific kinetics in this source; supports enzyme identity and pathway placement rather than quantitative physiology | Göbel et al., 2002, https://doi.org/10.1128/JB.184.1.216-223.2002 (gobel2002degradationofaromatics pages 5-5) |
Table: This table compiles the main functional, genetic, biochemical, and systems-level evidence for PcaF in Pseudomonas putida KT2440. It highlights the confirmed thiolase reaction, pathway role in β-ketoadipate catabolism, and how recent lignin-valorization studies contextualize the importance of the pathway around PcaF.
Werner et al. provide a pathway schematic illustrating the engineered block at pcaIJ for β‑ketoadipate accumulation and bioreactor data showing titers and productivity over time (werner2023ligninconversionto media ac5a69ec, werner2023ligninconversionto media 88d7ff26).
References
(nogales2007characterizationofthe pages 6-7): Juan Nogales, Raffaella Macchi, Federico Franchi, Dagania Barzaghi, Cristina Fernández, José L García, Giovanni Bertoni, and Eduardo Díaz. Characterization of the last step of the aerobic phenylacetic acid degradation pathway. Microbiology, 153 Pt 2:357-65, Feb 2007. URL: https://doi.org/10.1099/mic.0.2006/002444-0, doi:10.1099/mic.0.2006/002444-0. This article has 78 citations and is from a peer-reviewed journal.
(nogales2007characterizationofthe pages 4-5): Juan Nogales, Raffaella Macchi, Federico Franchi, Dagania Barzaghi, Cristina Fernández, José L García, Giovanni Bertoni, and Eduardo Díaz. Characterization of the last step of the aerobic phenylacetic acid degradation pathway. Microbiology, 153 Pt 2:357-65, Feb 2007. URL: https://doi.org/10.1099/mic.0.2006/002444-0, doi:10.1099/mic.0.2006/002444-0. This article has 78 citations and is from a peer-reviewed journal.
(werner2023ligninconversionto pages 1-2): Allison Z. Werner, William T. Cordell, Ciaran W. Lahive, Bruno C. Klein, Christine A. Singer, Eric C. D. Tan, Morgan A. Ingraham, Kelsey J. Ramirez, Dong Hyun Kim, Jacob Nedergaard Pedersen, Christopher W. Johnson, Brian F. Pfleger, Gregg T. Beckham, and Davinia Salvachúa. Lignin conversion to β-ketoadipic acid by pseudomonas putida via metabolic engineering and bioprocess development. Science Advances, Sep 2023. URL: https://doi.org/10.1126/sciadv.adj0053, doi:10.1126/sciadv.adj0053. This article has 82 citations and is from a highest quality peer-reviewed journal.
(nogales2007characterizationofthe pages 2-4): Juan Nogales, Raffaella Macchi, Federico Franchi, Dagania Barzaghi, Cristina Fernández, José L García, Giovanni Bertoni, and Eduardo Díaz. Characterization of the last step of the aerobic phenylacetic acid degradation pathway. Microbiology, 153 Pt 2:357-65, Feb 2007. URL: https://doi.org/10.1099/mic.0.2006/002444-0, doi:10.1099/mic.0.2006/002444-0. This article has 78 citations and is from a peer-reviewed journal.
(borchert2024machinelearninganalysis pages 6-7): Andrew J. Borchert, Alissa C. Bleem, Hyun Gyu Lim, Kevin Rychel, Keven D. Dooley, Zoe A. Kellermyer, Tracy L. Hodges, Bernhard O. Palsson, and Gregg T. Beckham. Machine learning analysis of rb-tnseq fitness data predicts functional gene modules in pseudomonas putida kt2440. Mar 2024. URL: https://doi.org/10.1128/msystems.00942-23, doi:10.1128/msystems.00942-23. This article has 10 citations and is from a peer-reviewed journal.
(gobel2002degradationofaromatics pages 1-2): Markus Göbel, Kerstin Kassel-Cati, Eberhard Schmidt, and Walter Reineke. Degradation of aromatics and chloroaromatics by pseudomonas sp. strain b13: cloning, characterization, and analysis of sequences encoding 3-oxoadipate:succinyl-coenzyme a (coa) transferase and 3-oxoadipyl-coa thiolase. Journal of Bacteriology, 184:216-223, Jan 2002. URL: https://doi.org/10.1128/jb.184.1.216-223.2002, doi:10.1128/jb.184.1.216-223.2002. This article has 38 citations and is from a peer-reviewed journal.
(werner2023ligninconversionto pages 2-4): Allison Z. Werner, William T. Cordell, Ciaran W. Lahive, Bruno C. Klein, Christine A. Singer, Eric C. D. Tan, Morgan A. Ingraham, Kelsey J. Ramirez, Dong Hyun Kim, Jacob Nedergaard Pedersen, Christopher W. Johnson, Brian F. Pfleger, Gregg T. Beckham, and Davinia Salvachúa. Lignin conversion to β-ketoadipic acid by pseudomonas putida via metabolic engineering and bioprocess development. Science Advances, Sep 2023. URL: https://doi.org/10.1126/sciadv.adj0053, doi:10.1126/sciadv.adj0053. This article has 82 citations and is from a highest quality peer-reviewed journal.
(werner2023ligninconversionto media ac5a69ec): Allison Z. Werner, William T. Cordell, Ciaran W. Lahive, Bruno C. Klein, Christine A. Singer, Eric C. D. Tan, Morgan A. Ingraham, Kelsey J. Ramirez, Dong Hyun Kim, Jacob Nedergaard Pedersen, Christopher W. Johnson, Brian F. Pfleger, Gregg T. Beckham, and Davinia Salvachúa. Lignin conversion to β-ketoadipic acid by pseudomonas putida via metabolic engineering and bioprocess development. Science Advances, Sep 2023. URL: https://doi.org/10.1126/sciadv.adj0053, doi:10.1126/sciadv.adj0053. This article has 82 citations and is from a highest quality peer-reviewed journal.
(werner2023ligninconversionto media 88d7ff26): Allison Z. Werner, William T. Cordell, Ciaran W. Lahive, Bruno C. Klein, Christine A. Singer, Eric C. D. Tan, Morgan A. Ingraham, Kelsey J. Ramirez, Dong Hyun Kim, Jacob Nedergaard Pedersen, Christopher W. Johnson, Brian F. Pfleger, Gregg T. Beckham, and Davinia Salvachúa. Lignin conversion to β-ketoadipic acid by pseudomonas putida via metabolic engineering and bioprocess development. Science Advances, Sep 2023. URL: https://doi.org/10.1126/sciadv.adj0053, doi:10.1126/sciadv.adj0053. This article has 82 citations and is from a highest quality peer-reviewed journal.
(werner2023ligninconversionto pages 8-10): Allison Z. Werner, William T. Cordell, Ciaran W. Lahive, Bruno C. Klein, Christine A. Singer, Eric C. D. Tan, Morgan A. Ingraham, Kelsey J. Ramirez, Dong Hyun Kim, Jacob Nedergaard Pedersen, Christopher W. Johnson, Brian F. Pfleger, Gregg T. Beckham, and Davinia Salvachúa. Lignin conversion to β-ketoadipic acid by pseudomonas putida via metabolic engineering and bioprocess development. Science Advances, Sep 2023. URL: https://doi.org/10.1126/sciadv.adj0053, doi:10.1126/sciadv.adj0053. This article has 82 citations and is from a highest quality peer-reviewed journal.
(gobel2002degradationofaromatics pages 5-5): Markus Göbel, Kerstin Kassel-Cati, Eberhard Schmidt, and Walter Reineke. Degradation of aromatics and chloroaromatics by pseudomonas sp. strain b13: cloning, characterization, and analysis of sequences encoding 3-oxoadipate:succinyl-coenzyme a (coa) transferase and 3-oxoadipyl-coa thiolase. Journal of Bacteriology, 184:216-223, Jan 2002. URL: https://doi.org/10.1128/jb.184.1.216-223.2002, doi:10.1128/jb.184.1.216-223.2002. This article has 38 citations and is from a peer-reviewed journal.
UniProt accession Q88N39 corresponds to pcaF-I / PP_1377 in Pseudomonas putida KT2440 and is annotated as beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), the enzyme that catalyzes thiolytic cleavage of beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA in the beta-ketoadipate pathway [file:PSEPK/pcaF/pcaF-uniprot.txt "Name=pcaF-I"; "Catalyzes thiolytic cleavage of beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA"; "beta-ketoadipate pathway; acetyl-CoA and succinyl-CoA from 3-oxoadipate: step 2/2."].
KT2440 proteomics supports placement of PcaF in convergent aromatic catabolism: PcaF was induced during growth on benzoate, p-hydroxybenzoate, and vanillin, consistent with use in the shared beta-ketoadipate degradation pathway [PMID:16470664 Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis, "beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway."].
Older genetics in P. putida PRS2000 identifies pcaF as the terminal thiolase step of the pathway [PMID:7961399 Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate, "pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway."].
pcaF expression is coordinated with the protocatechuate branch regulator PcaR and induced by beta-ketoadipate [PMID:8522507 Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida beta-ketoadipate pathway, "PcaR, a transcriptional activator of several genes of the beta-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate beta-ketoadipate induces both genes."].
The KT2440 structural paper treats PcaF as a degradative thiolase model and shows features expected for degradative rather than biosynthetic thiolases [PMID:32647822 Structural basis for differentiation between two classes of thiolase: Degradative vs biosynthetic thiolase, "we exploit, a tetrameric degradative thiolase from Pseudomonas putida KT2440 annotated as PcaF, as a model system"; "Degradative thiolases have different active site architecture when compared to biosynthetic thiolases"].
Current GOA for Q88N39 is entirely automated and contains six annotations. The most specific molecular function term is GO:0033812 3-oxoadipyl-CoA thiolase activity; the two generic acyltransferase terms are redundant umbrella labels, and GO:0006629 lipid metabolic process looks like a family-level over-annotation rather than a pathway-specific assignment for this aromatic catabolic enzyme [file:PSEPK/pcaF/pcaF-goa.tsv "GO:0033812"; "GO:0016746"; "GO:0016747"; "GO:0006629"; "GO:0019619"].
id: Q88N39
gene_symbol: pcaF-I
product_type: PROTEIN
aliases:
- pcaF
- PP_1377
status: DRAFT
taxon:
id: NCBITaxon:160488
label: Pseudomonas putida KT2440
description: Beta-ketoadipyl-CoA thiolase of the protocatechuate branch of the beta-ketoadipate pathway.
PcaF-I catalyzes thiolytic cleavage of 3-oxoadipyl-CoA to succinyl-CoA and acetyl-CoA,
completing assimilation of 3,4-dihydroxybenzoate-derived carbon into central metabolism.
Available evidence supports a degradative thiolase specialized for aromatic compound
catabolism rather than a general lipid-metabolism enzyme.
existing_annotations:
- term:
id: GO:0003988
label: acetyl-CoA C-acyltransferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000117
review:
summary: Correct as a broad parent activity for a thiolase, but it is less informative
than the specific 3-oxoadipyl-CoA thiolase term already present.
action: KEEP_AS_NON_CORE
reason: PcaF-I is a C-acyltransferase/thiolase, but the curated core function should
use GO:0033812 to capture substrate specificity.
- term:
id: GO:0016746
label: acyltransferase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: Too general to be useful for this gene and likely inherited from family-level
domain mappings rather than pathway-specific evidence.
action: MARK_AS_OVER_ANNOTATED
reason: The specific thiolase activity is already represented by GO:0033812, so this
broad umbrella term adds little biological information.
- term:
id: GO:0016747
label: acyltransferase activity, transferring groups other than amino-acyl groups
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: Mechanistically true at a very high level, but still a redundant broad family
term relative to the specific 3-oxoadipyl-CoA thiolase annotation.
action: MARK_AS_OVER_ANNOTATED
reason: Pathway-specific curation should prefer the substrate-resolved thiolase term.
- term:
id: GO:0033812
label: 3-oxoadipyl-CoA thiolase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: Best current molecular function term for PcaF-I and consistent with UniProt
pathway assignment, classical pcaF genetics, KT2440 proteomics, and the KT2440 structural
degradative-thiolase paper.
action: ACCEPT
reason: This term captures the specific beta-ketoadipyl-CoA/3-oxoadipyl-CoA thiolase
activity that defines the enzyme.
- term:
id: GO:0006629
label: lipid metabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000117
review:
summary: Not supported as a core role for this gene. The enzyme functions in aromatic
compound degradation through the beta-ketoadipate pathway, not in canonical lipid
metabolism.
action: REMOVE
reason: This appears to be a family-level over-projection from thiolase chemistry rather
than a pathway-specific assignment for PcaF-I.
- term:
id: GO:0019619
label: 3,4-dihydroxybenzoate catabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: Appropriate biological-process annotation because PcaF-I acts in the shared
beta-ketoadipate segment used to complete protocatechuate/3,4-dihydroxybenzoate degradation.
action: ACCEPT
reason: Proteomics and classical pca pathway genetics support participation of PcaF in
the protocatechuate branch of aromatic compound catabolism.
core_functions:
- description: PcaF-I is a degradative thiolase that cleaves 3-oxoadipyl-CoA to acetyl-CoA
and succinyl-CoA in the beta-ketoadipate pathway, supporting assimilation of 3,4-dihydroxybenzoate-derived
carbon into central metabolism.
molecular_function:
id: GO:0033812
label: 3-oxoadipyl-CoA thiolase activity
directly_involved_in:
- id: GO:0019619
label: 3,4-dihydroxybenzoate catabolic process
references:
- id: GO_REF:0000002
title: "Gene Ontology annotation through association of InterPro records with GO terms."
findings: []
- id: GO_REF:0000117
title: "Electronic Gene Ontology annotations created by ARBA machine learning models"
findings: []
- id: GO_REF:0000120
title: "Combined Automated Annotation using Multiple IEA Methods."
findings: []
- id: PMID:7961399
title: "Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate"
findings: []
- id: PMID:8522507
title: "Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida beta-ketoadipate pathway"
findings: []
- id: PMID:12534463
title: "Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440"
findings: []
- id: PMID:16470664
title: "Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis"
findings: []
- id: PMID:26913973
title: "The revisited genome of Pseudomonas putida KT2440 enlightens its value as a robust metabolic chassis"
findings: []
- id: PMID:32647822
title: "Structural basis for differentiation between two classes of thiolase: Degradative vs biosynthetic thiolase"
findings: []
- id: file:PSEPK/pcaF/pcaF-deep-research-falcon.md
title: "Falcon deep research report for pcaF-I in Pseudomonas putida KT2440"
findings: []
- id: file:PSEPK/pcaF/pcaF-notes.md
title: "Curator notes for pcaF-I in Pseudomonas putida KT2440"
findings: []
proposed_new_terms: []
suggested_questions:
- question: Is PcaF-I dedicated to beta-ketoadipate turnover in vivo, or can it efficiently
process alternative CoA thioesters under physiological conditions?
- question: How is pcaF-I coordinated transcriptionally with the rest of the protocatechuate
branch in KT2440 under mixed aromatic substrates?
suggested_experiments:
- description: Construct a clean pcaF-I deletion in KT2440 and test growth plus metabolite
accumulation on protocatechuate, p-hydroxybenzoate, vanillate, and benzoate to confirm
the pathway block at the 3-oxoadipyl-CoA cleavage step.
hypothesis: Loss of pcaF-I will prevent efficient flux through the beta-ketoadipate pathway
and cause accumulation of upstream protocatechuate-branch intermediates.
- description: Purify PcaF-I and compare catalytic efficiency for 3-oxoadipyl-CoA versus
alternative acyl-CoA substrates to quantify whether the enzyme is pathway-specialized
or broadly permissive like other degradative thiolases.
hypothesis: PcaF-I will show clear catalytic preference for 3-oxoadipyl-CoA despite sharing
general thiolase-family chemistry with broader acyltransferases.