aroK

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0000287 magnesium ion binding	IEA GO_REF:0000104	ACCEPT	Summary: Shikimate kinase requires a divalent Mg(2+) cofactor for catalysis; UniProt records binding of one Mg(2+) ion per subunit, coordinated near the ATP-binding P-loop. This is a well-supported, family-conserved molecular function. Reason: Magnesium binding is intrinsic to shikimate kinase catalysis and is supported by the HAMAP rule and conserved metal-binding residue; consistent with the enzymology of the shikimate kinase family.
GO:0004765 shikimate kinase activity	IEA GO_REF:0000120	ACCEPT	Summary: This is the core molecular function of AroK, supported by the EC 2.7.1.71 assignment, the RHEA:13121 catalytic-activity mapping, HAMAP rule MF_00109, and strong family/domain evidence (Pfam SKI, shikimate kinase signature). Reason: Directly represents the defining enzymatic activity of the gene product and is the central core function.
GO:0005524 ATP binding	IEA GO_REF:0000104	ACCEPT	Summary: AroK is an ATP-dependent kinase that uses ATP as the phosphoryl donor; it contains a P-loop/Walker A motif (residues 11-16) and additional ATP-contacting residues. ATP binding is a required, well-supported molecular function. Reason: ATP binding is an obligatory part of the shikimate kinase reaction and is supported by the conserved P-loop nucleotide-binding fold.
GO:0005737 cytoplasm	IEA GO_REF:0000120	ACCEPT	Summary: AroK is a soluble cytoplasmic enzyme acting in central metabolism, consistent with UniProt subcellular location and the general biology of bacterial shikimate-pathway enzymes. Reason: Correct localization for a cytoplasmic biosynthetic enzyme; consistent with the cytosol annotation below.
GO:0005829 cytosol	IEA GO_REF:0000118	ACCEPT	Summary: TreeGrafter assigns the cytosol component, the more specific child of cytoplasm, consistent with a soluble bacterial shikimate kinase. Reason: Accurate and slightly more specific localization than GO:0005737; both are appropriate for this enzyme.
GO:0009423 chorismate biosynthetic process	IEA GO_REF:0000120	ACCEPT	Summary: AroK catalyzes step 5 of 7 in chorismate biosynthesis (the shikimate pathway), converting shikimate to shikimate 3-phosphate en route to chorismate. This is the precise biological process for the gene. Reason: Directly and specifically captures the pathway role of AroK; UniPathway UPA00053/UER00088 corroborates the chorismate-biosynthesis placement.

Core Functions

ATP- and Mg(2+)-dependent shikimate kinase that phosphorylates the 3-hydroxyl of shikimate to form shikimate 3-phosphate, catalyzing step 5 of the shikimate pathway in chorismate biosynthesis.

Molecular Function:

shikimate kinase activity

Directly Involved In:

chorismate biosynthetic process

Supporting Evidence:

GO_REF:0000120
EC 2.7.1.71; RHEA:13121 shikimate + ATP = 3-phosphoshikimate + ADP + H(+); HAMAP-Rule MF_00109.

References

GO_REF:0000104

Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features

GO_REF:0000118

TreeGrafter-generated GO annotations

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:12534463

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440.

Suggested Experiments

Experiment: Determine steady-state kinetic parameters (Km/kcat for shikimate and ATP, Mg(2+) dependence) of recombinant KT2440 AroK to confirm substrate specificity and rule out promiscuous quinate kinase activity.

Experiment: Assess essentiality/fitness of an aroK deletion in KT2440 on minimal medium with and without aromatic amino acid supplementation to test for aromatic auxotrophy.

Deep Research

Falcon

(aroK-deep-research-falcon.md)

this with annotations you find in gene/protein databases, but these can be outdated or inaccurate. Falcon Edison Scientific Literature 23 citations 2 artifacts 2026-06-11T21:09:29.533954

Edison artifact artifact-00 (text/markdown)

## Context ID: pqac-00000016 The engineered Pseudomonas putida KT2440 strains and their genotypes, including those with the specified modifications (e.g., ΔpykF (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 aroK (UniProt Q88CV1, locus PP_5079) in Pseudomonas putida KT2440

0) Gene/protein identity verification (critical)

The target provided (UniProt Q88CV1; gene aroK; ordered locus PP_5079) is annotated as shikimate kinase (SK), EC 2.7.1.71, belonging to the shikimate kinase family with P-loop NTPase/“shikimate/glucono_kinase” domain architecture (per user-supplied UniProt context). In the retrieved literature, aroK consistently refers to a bacterial shikimate kinase I isoenzyme (contrasted with aroL, shikimate kinase II), consistent with the UniProt description and EC assignment. (gu2016tunableswitchmediated pages 1-2, niraula2025aromaticaminoacids pages 8-10)

Limitation: none of the retrieved papers explicitly cross-references UniProt accession Q88CV1 or the KT2440 locus tag PP_5079 in the text excerpts obtained, so KT2440-specific details below are supported by P. putida KT2440 studies that manipulate aroK at the gene level, combined with general shikimate-kinase enzymology. (ling2022muconicacidproduction pages 2-3, gu2016tunableswitchmediated pages 1-2)

1) Key concepts and definitions (current understanding)

1.1 Shikimate pathway and the biochemical role of AroK

The shikimate pathway is the central biosynthetic route from central carbon metabolism toward chorismate, a key branch point precursor for aromatic amino acids and numerous aromatic metabolites. Within this pathway, shikimate kinase (SK; EC 2.7.1.71) catalyzes the ATP-dependent phosphorylation of shikimate to shikimate-3-phosphate (S3P); this step is required to proceed to 5-enolpyruvyl-shikimate-3-phosphate (EPSP) and ultimately chorismate. (niraula2025aromaticaminoacids pages 8-10)

Reaction (core functional annotation):
- Shikimate + ATP → Shikimate-3-phosphate + ADP (SK; EC 2.7.1.71). (niraula2025aromaticaminoacids pages 8-10)

1.2 AroK versus AroL (bacterial isoenzymes)

Many bacteria encode two shikimate kinase isoenzymes:
- AroK: shikimate kinase I
- AroL: shikimate kinase II

This redundancy is important in both physiology and metabolic engineering: engineered “shikimate-overproducer” strains often inactivate/decrease shikimate kinase activity to accumulate shikimate upstream, but complete loss of SK activity can lead to aromatic auxotrophy (requiring supplementation with aromatic compounds). (gu2016tunableswitchmediated pages 1-2)

1.3 Structure/function concepts relevant to substrate specificity

While organism-specific kinetics for P. putida KT2440 AroK were not retrieved in the accessible excerpts, a recent synthesis of bacterial SK structure–function notes that shikimate-binding can be described via subsites (CX, OCORE, OLID) and that specific conserved residues (e.g., positions corresponding to R60 and R140 in one referenced mapping) are functionally critical; mutating strictly conserved residues severely compromises function in engineering contexts, consistent with strong substrate-enzyme specificity requirements for shikimate recognition. (bo2023multiplemetabolicengineering pages 7-9)

2) Functional annotation for P. putida KT2440 aroK (Q88CV1)

2.1 Molecular function (enzyme activity)

AroK is a shikimate kinase (SK; EC 2.7.1.71) catalyzing the ATP-dependent phosphorylation of shikimate to shikimate-3-phosphate in the shikimate pathway. (niraula2025aromaticaminoacids pages 8-10, gu2016tunableswitchmediated pages 1-2)

2.2 Biological process / pathway placement

AroK functions in chorismate biosynthesis via the shikimate pathway, enabling downstream synthesis of aromatic amino acids and diverse chorismate-derived metabolites. (niraula2025aromaticaminoacids pages 8-10)

2.3 Cellular localization (where it acts)

The retrieved sources explicitly contrast chloroplast-localized shikimate kinase isoforms in algae/plants with bacterial SK functioning without organellar targeting; thus, for bacteria including P. putida KT2440, the working assumption supported by this context is that SK is a soluble, intracellular (cytosolic) enzyme acting in central metabolism. (niraula2025aromaticaminoacids pages 8-10)

Limitation: no KT2440-specific localization experiment (e.g., fractionation, fluorescence tagging) was found in the retrieved set.

3) Recent developments and latest research (prioritizing 2023–2024)

3.1 KT2440 as an aromatics/synbio chassis (expert synthesis, 2024)

A 2024 authoritative review/perspective describes P. putida KT2440’s rise as a synthetic biology chassis and emphasizes that its central metabolism delivers high NADPH regeneration (via ED/EDEMP-related architecture), supporting expression of heterologous pathways and tolerance to stress—properties relevant to building shikimate-pathway-derived aromatic production strains. (lorenzo2024pseudomonasputidakt2440 pages 2-4, lorenzo2024pseudomonasputidakt2440 pages 4-7, lorenzo2024pseudomonasputidakt2440 pages 1-2)

This review also highlights KT2440’s native and engineered capabilities in aromatic metabolism and production, citing examples of engineered production of shikimate-derived aromatics such as phenol, vanillate, and anthranilate. (lorenzo2024pseudomonasputidakt2440 pages 4-7)

Source: de Lorenzo et al., 2024-07, Journal of Bacteriology. https://doi.org/10.1128/jb.00136-24 (lorenzo2024pseudomonasputidakt2440 pages 2-4)

3.2 2024 “shikimate pathway-dependent catabolism” (SDC) rewiring to near-theoretical yields

A 2024 study reports a major conceptual advance: reprogramming P. putida so that growth-supporting catabolism becomes dependent on shikimate-pathway-derived reactions, demonstrating substantial metabolic plasticity and enabling very high-yield aromatic production. They report 89% of the maximum theoretical yield for 4-hydroxybenzoate in minimal medium and describe adaptive evolution mutations (e.g., in miaA and mexT) as important contributors. (santos2024shikimatepathwaydependentcatabolism pages 5-7)

Source: dos Santos et al., 2024-08 (preprint server posting). https://doi.org/10.21203/rs.3.rs-4761679/v1 (santos2024shikimatepathwaydependentcatabolism pages 5-7)

3.3 2024 combinatorial pathway engineering to find bottlenecks (pABA)

A 2024 design-of-experiments/combinatorial expression study in P. putida quantitatively mapped how varying shikimate- and pABA-pathway gene expression influences production. It reports wide performance spread (2–186.2 mg/L) and improved strains (~232.1 mg/L), identifying aroB (not aroK) as a key bottleneck for pABA production under tested conditions, while situating the shikimate pathway as a major industrial node (muconate, tryptophan, salicylic acid, vanillin, 2-phenylethanol). (camposmagana2024combinatorialengineeringreveals pages 1-4)

Source: Campos-Magaña et al., 2024-06 (bioRxiv). https://doi.org/10.1101/2024.06.17.599342 (camposmagana2024combinatorialengineeringreveals pages 1-4)

4) Current applications and real-world implementations (with P. putida examples)

4.1 AroK as a practical flux-control lever in KT2440 muconate bioproduction

A key KT2440 implementation is cis,cis-muconate production, a bioprivileged platform chemical. In a high-impact Nature Communications study, KT2440-derived strains were engineered with aroK under the inducible Ptac promoter, either alone (ΔpykF::Ptac:aroK) or together with aroB (ΔpykF::Ptac:aroK:aroB), demonstrating that modulating aroK expression is used in practice to tune shikimate-pathway flux in KT2440-derived production strains. (ling2022muconicacidproduction pages 2-3, ling2022muconicacidproduction media b6590159)

The same study reports 33.7 g/L muconate, 0.18 g/L/h productivity, and 46% molar yield (reported as 92% of maximum theoretical yield) in the rationally engineered strain context. (ling2022muconicacidproduction pages 2-3)

Source: Ling et al., 2022-08, Nature Communications. https://doi.org/10.1038/s41467-022-32296-y (ling2022muconicacidproduction pages 2-3)

4.2 KT2440 production of gallic acid from glycerol (2023)

A 2023 study demonstrates conversion of glycerol to the antioxidant gallic acid in KT2440 by combining heterologous pathway expression with deletions that block product degradation, explicitly leveraging KT2440’s metabolic and chassis advantages. The reported titer is 346.7 ± 0.004 mg/L gallic acid after 72 h (shake flasks). (dias2023fromdegraderto pages 1-2)

Source: Dias et al., 2023-11, International Microbiology. https://doi.org/10.1007/s10123-022-00282-5 (dias2023fromdegraderto pages 1-2)

5) Expert opinions and analysis (authoritative sources)

5.1 Why KT2440 is repeatedly chosen for shikimate-pathway aromatics

Across authoritative sources, KT2440 is characterized as an “industriphilic” synthetic biology chassis and “versatile aromatics cell factory,” with repeated emphasis on:
- High NADPH regeneration and redox-biased central metabolism, supporting reductive biosynthesis and heterologous pathway operation. (lorenzo2024pseudomonasputidakt2440 pages 2-4, lorenzo2024pseudomonasputidakt2440 pages 4-7)
- Intrinsic tolerance to aromatic compounds and possession of oxygenase repertoires to transform them—useful because many aromatics are toxic or require harsh processes. (lorenzo2024pseudomonasputidakt2440 pages 4-7)

These points contextualize aroK’s importance: as a control point in the shikimate pathway, aroK expression/activity influences whether carbon proceeds toward chorismate-derived products (desired aromatics) or accumulates upstream intermediates (e.g., shikimate) (gu2016tunableswitchmediated pages 1-2, ling2022muconicacidproduction pages 2-3).

6) Quantitative statistics and data points from recent and relevant studies

Key quantitative outcomes tied to shikimate-pathway engineering in P. putida include:
- Muconate production (KT2440): 33.7 g/L, 0.18 g/L/h, 46% molar yield (92% of max theoretical); includes strains engineered with Ptac:aroK (flux control through shikimate pathway). (ling2022muconicacidproduction pages 2-3, ling2022muconicacidproduction media b6590159)
- Gallic acid production (KT2440): 346.7 ± 0.004 mg/L after 72 h from 10 g/L glycerol in shake flasks (as described in excerpt). (dias2023fromdegraderto pages 1-2)
- pABA production (P. putida, 2024 DoE): 2–186.2 mg/L across sampled genotypes; improved to ~232.1 mg/L; identified aroB as bottleneck. (camposmagana2024combinatorialengineeringreveals pages 1-4)
- 4-hydroxybenzoate yield (P. putida, 2024 SDC concept): 89% of maximum theoretical yield in minimal medium (yield benchmark claim). (santos2024shikimatepathwaydependentcatabolism pages 5-7)

7) Evidence summary table

Claim/Topic	Evidence summary	Organism/context	Quantitative data	Source (with year, DOI URL)
Core enzymatic reaction of shikimate kinase	Shikimate kinase (SK; EC 2.7.1.71) phosphorylates shikimate at the C3 hydroxyl using ATP to form shikimate-3-phosphate (S3P), a required step toward EPSP and chorismate in the shikimate pathway (niraula2025aromaticaminoacids pages 8-10)	General bacterial/plant/algal shikimate-pathway context	Reaction: shikimate + ATP -> shikimate-3-phosphate + ADP	Niraula et al., 2025, https://doi.org/10.3390/biotech14010006
Bacterial isoenzymes AroK and AroL	In bacteria, two shikimate kinase isoenzymes are commonly recognized: shikimate kinase I (AroK) and shikimate kinase II (AroL). Engineering studies often repress/delete these enzymes to accumulate shikimate, but full loss causes auxotrophy and necessitates aromatic supplementation (gu2016tunableswitchmediated pages 1-2)	Engineered bacterial shikimate producers, especially E. coli	Example reported for engineered E. coli: 13.15 g/L shikimate in 5-L fed-batch using tunable aroK expression rather than permanent deletion	Gu et al., 2016, https://doi.org/10.1038/srep29745
Shikimate-binding determinants in AroK	Structural/functional analysis of bacterial AroK identifies shikimate-binding subsites CX, OCORE, and OLID. Conserved residues contacting the substrate include positions corresponding to R60 and R140; mutating these severely impaired function/growth in engineering contexts (bo2023multiplemetabolicengineering pages 7-9)	Bacterial AroK, with cited structural work including Helicobacter pylori and other bacterial SKs	No single kinetic value in snippet; severe growth inhibition observed for key conserved-site mutants	Bo et al., 2023, https://doi.org/10.3390/metabo13060747
KT2440-specific aroK manipulation in muconate engineering	In Pseudomonas putida KT2440-derived strains for muconate production, aroK was deliberately overexpressed from Ptac, either alone or together with aroB (e.g., ΔpykF::Ptac:aroK and ΔpykF::Ptac:aroK:aroB), indicating aroK is a practical flux-control point in the native shikimate pathway (ling2022muconicacidproduction pages 2-3)	Pseudomonas putida KT2440 metabolic engineering for muconic acid	Muconate process in study reached 33.7 g/L muconate, 0.18 g/L/h, 46% molar yield (92% of maximum theoretical yield); snippet specifically highlights Ptac:aroK strain designs	Ling et al., 2022, https://doi.org/10.1038/s41467-022-32296-y
2024 pABA combinatorial engineering identifies pathway bottleneck	A 2024 DoE study in P. putida varied shikimate- and pABA-pathway gene expression across 14 representative strains sampled from 512 possible combinations. The analysis identified aroB, not aroK, as a significant bottleneck for pABA production, while noting aroK overexpression had been beneficial in related production contexts (camposmagana2024combinatorialengineeringreveals pages 1-4)	Pseudomonas putida shikimate/pABA pathway engineering	Titers ranged from 2 to 186.2 mg/L initially; second-round designs reached ~232.1 mg/L	Campos-Magaña et al., 2024, https://doi.org/10.1101/2024.06.17.599342
2024 shikimate pathway-dependent catabolism (SDC)	A 2024 P. putida study rewired metabolism so shikimate-pathway-derived reactions supplied pyruvate, demonstrating strong metabolic plasticity of the pathway relevant to enzymes such as AroK that control flux into downstream aromatic intermediates (santos2024shikimatepathwaydependentcatabolism pages 5-7)	Pseudomonas putida engineered for aromatic production	89% of maximum theoretical yield for 4-hydroxybenzoate in minimal medium; adapted strains also produced salicylate and 3-hydroxybenzoate at high yields	dos Santos et al., 2024, https://doi.org/10.21203/rs.3.rs-4761679/v1
2023 real-world aromatic production application in KT2440	P. putida KT2440 was engineered to produce gallic acid from glycerol using a synthetic operon (aroG4, quiC, pobA*) plus deletions blocking degradation (pcaHG and galTAPR). This shows the practical biotechnological value of controlling shikimate-pathway flux in KT2440, even though aroK itself was not directly manipulated in the snippet (dias2023fromdegraderto pages 1-2)	Pseudomonas putida KT2440 producing gallic acid from glycerol	346.7 ± 0.004 mg/L gallic acid after 72 h in shake flasks	Dias et al., 2023, https://doi.org/10.1007/s10123-022-00282-5

Table: This table summarizes the most relevant gathered evidence for annotating Pseudomonas putida KT2440 aroK as shikimate kinase, including core reaction chemistry, bacterial isoenzyme context, and KT2440-specific engineering evidence. It is useful for separating direct functional evidence from broader pathway and application-level evidence.

8) Practical functional-annotation statement (recommended)

aroK (UniProt Q88CV1; PP_5079) encodes shikimate kinase (EC 2.7.1.71), a cytosolic ATP-dependent kinase that converts shikimate to shikimate-3-phosphate in the shikimate pathway, enabling downstream EPSP/chorismate formation and thereby aromatic amino acid and aromatic metabolite biosynthesis. (niraula2025aromaticaminoacids pages 8-10, gu2016tunableswitchmediated pages 1-2)

In P. putida KT2440, aroK is repeatedly treated as a flux-control point in industrially motivated shikimate-pathway engineering, including muconate production strains where aroK is overexpressed from Ptac in defined genomic backgrounds. (ling2022muconicacidproduction pages 2-3, ling2022muconicacidproduction media b6590159)

9) Gaps and recommended next evidence to retrieve (to strengthen annotation)

The current evidence set is strong for pathway placement and engineering relevance, but limited for P. putida KT2440-specific mechanistic details.

Most impactful missing items would be:
1) Direct biochemical characterization (Km/kcat for shikimate/ATP; Mg2+ dependence; substrate scope such as quinate) for KT2440 AroK.
2) Genetic essentiality/fitness measurements for KT2440 aroK under defined media (e.g., transposon insertion sequencing) and rescue by aromatic supplementation.
3) Subcellular localization experiments (fractionation or fluorescence tagging) in KT2440.

These gaps were not recoverable within the retrieved corpus and are therefore not asserted beyond supported inference.

References

(gu2016tunableswitchmediated pages 1-2): Pengfei Gu, Tianyuan Su, Qian Wang, Quanfeng Liang, and Qingsheng Qi. Tunable switch mediated shikimate biosynthesis in an engineered non-auxotrophic escherichia coli. Scientific Reports, Jul 2016. URL: https://doi.org/10.1038/srep29745, doi:10.1038/srep29745. This article has 43 citations and is from a peer-reviewed journal.
(niraula2025aromaticaminoacids pages 8-10): Archana Niraula, Amir Danesh, Natacha Merindol, Fatma Meddeb-Mouelhi, and Isabel Desgagné-Penix. Aromatic amino acids: exploring microalgae as a potential biofactory. BioTech, 14:6, Jan 2025. URL: https://doi.org/10.3390/biotech14010006, doi:10.3390/biotech14010006. This article has 9 citations.
(ling2022muconicacidproduction pages 2-3): Chen Ling, George L. Peabody, Davinia Salvachúa, Young-Mo Kim, Colin M. Kneucker, Christopher H. Calvey, Michela A. Monninger, Nathalie Munoz Munoz, Brenton C. Poirier, Kelsey J. Ramirez, Peter C. St. John, Sean P. Woodworth, Jon K. Magnuson, Kristin E. Burnum-Johnson, Adam M. Guss, Christopher W. Johnson, and Gregg T. Beckham. Muconic acid production from glucose and xylose in pseudomonas putida via evolution and metabolic engineering. Nature Communications, Aug 2022. URL: https://doi.org/10.1038/s41467-022-32296-y, doi:10.1038/s41467-022-32296-y. This article has 141 citations and is from a highest quality peer-reviewed journal.
(bo2023multiplemetabolicengineering pages 7-9): Taidong Bo, Chen Wu, Zeting Wang, Hao Jiang, Fei-Yong Wang, N. Chen, and Yanjun Li. Multiple metabolic engineering strategies to improve shikimate titer in escherichia coli. Metabolites, 13:747, Jun 2023. URL: https://doi.org/10.3390/metabo13060747, doi:10.3390/metabo13060747. This article has 12 citations.
(lorenzo2024pseudomonasputidakt2440 pages 2-4): Victor de Lorenzo, Danilo Pérez-Pantoja, and Pablo I. Nikel. pseudomonas putida kt2440: the long journey of a soil-dweller to become a synthetic biology chassis. Journal of Bacteriology, Jul 2024. URL: https://doi.org/10.1128/jb.00136-24, doi:10.1128/jb.00136-24. This article has 78 citations and is from a peer-reviewed journal.
(lorenzo2024pseudomonasputidakt2440 pages 4-7): Victor de Lorenzo, Danilo Pérez-Pantoja, and Pablo I. Nikel. pseudomonas putida kt2440: the long journey of a soil-dweller to become a synthetic biology chassis. Journal of Bacteriology, Jul 2024. URL: https://doi.org/10.1128/jb.00136-24, doi:10.1128/jb.00136-24. This article has 78 citations and is from a peer-reviewed journal.
(lorenzo2024pseudomonasputidakt2440 pages 1-2): Victor de Lorenzo, Danilo Pérez-Pantoja, and Pablo I. Nikel. pseudomonas putida kt2440: the long journey of a soil-dweller to become a synthetic biology chassis. Journal of Bacteriology, Jul 2024. URL: https://doi.org/10.1128/jb.00136-24, doi:10.1128/jb.00136-24. This article has 78 citations and is from a peer-reviewed journal.
(santos2024shikimatepathwaydependentcatabolism pages 5-7): Vitor Martins dos Santos, Lyon Bruinsma, Christos Batianis, Sara Moreno-Paz, Kesi Kurnia, Job Dirkmaat, Alexandra Müller, Jose Juncosa Nuñez, and Ruud Weusthuis. Shikimate pathway-dependent catabolism: enabling near-to-maximum production yield of aromatics. Unknown journal, Aug 2024. URL: https://doi.org/10.21203/rs.3.rs-4761679/v1, doi:10.21203/rs.3.rs-4761679/v1.
(camposmagana2024combinatorialengineeringreveals pages 1-4): Marco A Campos-Magaña, Sara Moreno-Paz, Vitor AP Martins dos Santos, Luis Garcia-Morales, and Maria Suarez-Diez. Combinatorial engineering reveals shikimate pathway bottlenecks in para-aminobenzoic acid production in pseudomonas putida. bioRxiv, Jun 2024. URL: https://doi.org/10.1101/2024.06.17.599342, doi:10.1101/2024.06.17.599342. This article has 0 citations.
(ling2022muconicacidproduction media b6590159): Chen Ling, George L. Peabody, Davinia Salvachúa, Young-Mo Kim, Colin M. Kneucker, Christopher H. Calvey, Michela A. Monninger, Nathalie Munoz Munoz, Brenton C. Poirier, Kelsey J. Ramirez, Peter C. St. John, Sean P. Woodworth, Jon K. Magnuson, Kristin E. Burnum-Johnson, Adam M. Guss, Christopher W. Johnson, and Gregg T. Beckham. Muconic acid production from glucose and xylose in pseudomonas putida via evolution and metabolic engineering. Nature Communications, Aug 2022. URL: https://doi.org/10.1038/s41467-022-32296-y, doi:10.1038/s41467-022-32296-y. This article has 141 citations and is from a highest quality peer-reviewed journal.
(dias2023fromdegraderto pages 1-2): Felipe M. S. Dias, Raoní K. Pantoja, José Gregório C. Gomez, and Luiziana F. Silva. From degrader to producer: reversing the gallic acid metabolism of pseudomonas putida kt2440. International Microbiology, 26:243-255, Nov 2023. URL: https://doi.org/10.1007/s10123-022-00282-5, doi:10.1007/s10123-022-00282-5. This article has 7 citations and is from a peer-reviewed journal.

Artifacts

Edison artifact artifact-00

Citations

niraula2025aromaticaminoacids pages 8-10
gu2016tunableswitchmediated pages 1-2
bo2023multiplemetabolicengineering pages 7-9
santos2024shikimatepathwaydependentcatabolism pages 5-7
camposmagana2024combinatorialengineeringreveals pages 1-4
ling2022muconicacidproduction pages 2-3
dias2023fromdegraderto pages 1-2
https://doi.org/10.1128/jb.00136-24
https://doi.org/10.21203/rs.3.rs-4761679/v1
https://doi.org/10.1101/2024.06.17.599342
https://doi.org/10.1038/s41467-022-32296-y
https://doi.org/10.1007/s10123-022-00282-5
https://doi.org/10.3390/biotech14010006
https://doi.org/10.1038/srep29745
https://doi.org/10.3390/metabo13060747
https://doi.org/10.1038/srep29745,
https://doi.org/10.3390/biotech14010006,
https://doi.org/10.1038/s41467-022-32296-y,
https://doi.org/10.3390/metabo13060747,
https://doi.org/10.1128/jb.00136-24,
https://doi.org/10.21203/rs.3.rs-4761679/v1,
https://doi.org/10.1101/2024.06.17.599342,
https://doi.org/10.1007/s10123-022-00282-5,

📄 View Raw YAML

id: Q88CV1
gene_symbol: aroK
product_type: PROTEIN
status: DRAFT
taxon:
  id: NCBITaxon:160488
  label: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950 / KT2440)
description: AroK is the shikimate kinase (EC 2.7.1.71) of Pseudomonas putida KT2440. It catalyzes the fifth step of the shikimate pathway, the ATP-dependent phosphorylation of the 3-hydroxyl group of shikimate to form shikimate 3-phosphate (3-phosphoshikimate), with release of ADP and a proton. The enzyme requires a Mg(2+) cofactor (one ion bound per subunit) and adopts the P-loop (Walker A) NTPase fold characteristic of the shikimate kinase family, with a glycine-rich nucleotide-binding loop near the N-terminus. As a cytoplasmic monomer, AroK provides shikimate 3-phosphate as the substrate for the downstream EPSP synthase (AroA) and ultimately chorismate, the branch-point precursor for aromatic amino acids (Phe, Tyr, Trp), folate, ubiquinone, and other aromatic metabolites. In bacteria, shikimate kinase activity can be encoded by two isoenzymes, AroK (shikimate kinase I) and AroL (shikimate kinase II); this step is a recognized flux-control point in shikimate-pathway metabolic engineering.
existing_annotations:
- term:
    id: GO:0000287
    label: magnesium ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000104
  qualifier: enables
  review:
    summary: Shikimate kinase requires a divalent Mg(2+) cofactor for catalysis; UniProt records binding of one Mg(2+) ion per subunit, coordinated near the ATP-binding P-loop. This is a well-supported, family-conserved molecular function.
    action: ACCEPT
    reason: Magnesium binding is intrinsic to shikimate kinase catalysis and is supported by the HAMAP rule and conserved metal-binding residue; consistent with the enzymology of the shikimate kinase family.
- term:
    id: GO:0004765
    label: shikimate kinase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: enables
  review:
    summary: This is the core molecular function of AroK, supported by the EC 2.7.1.71 assignment, the RHEA:13121 catalytic-activity mapping, HAMAP rule MF_00109, and strong family/domain evidence (Pfam SKI, shikimate kinase signature).
    action: ACCEPT
    reason: Directly represents the defining enzymatic activity of the gene product and is the central core function.
- term:
    id: GO:0005524
    label: ATP binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000104
  qualifier: enables
  review:
    summary: AroK is an ATP-dependent kinase that uses ATP as the phosphoryl donor; it contains a P-loop/Walker A motif (residues 11-16) and additional ATP-contacting residues. ATP binding is a required, well-supported molecular function.
    action: ACCEPT
    reason: ATP binding is an obligatory part of the shikimate kinase reaction and is supported by the conserved P-loop nucleotide-binding fold.
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: located_in
  review:
    summary: AroK is a soluble cytoplasmic enzyme acting in central metabolism, consistent with UniProt subcellular location and the general biology of bacterial shikimate-pathway enzymes.
    action: ACCEPT
    reason: Correct localization for a cytoplasmic biosynthetic enzyme; consistent with the cytosol annotation below.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IEA
  original_reference_id: GO_REF:0000118
  qualifier: located_in
  review:
    summary: TreeGrafter assigns the cytosol component, the more specific child of cytoplasm, consistent with a soluble bacterial shikimate kinase.
    action: ACCEPT
    reason: Accurate and slightly more specific localization than GO:0005737; both are appropriate for this enzyme.
- term:
    id: GO:0009423
    label: chorismate biosynthetic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  qualifier: involved_in
  review:
    summary: AroK catalyzes step 5 of 7 in chorismate biosynthesis (the shikimate pathway), converting shikimate to shikimate 3-phosphate en route to chorismate. This is the precise biological process for the gene.
    action: ACCEPT
    reason: Directly and specifically captures the pathway role of AroK; UniPathway UPA00053/UER00088 corroborates the chorismate-biosynthesis placement.
core_functions:
- description: ATP- and Mg(2+)-dependent shikimate kinase that phosphorylates the 3-hydroxyl of shikimate to form shikimate 3-phosphate, catalyzing step 5 of the shikimate pathway in chorismate biosynthesis.
  molecular_function:
    id: GO:0004765
    label: shikimate kinase activity
  supported_by:
  - reference_id: GO_REF:0000120
    supporting_text: EC 2.7.1.71; RHEA:13121 shikimate + ATP = 3-phosphoshikimate + ADP + H(+); HAMAP-Rule MF_00109.
  directly_involved_in:
  - id: GO:0009423
    label: chorismate biosynthetic process
proposed_new_terms: []
suggested_questions:
- question: Does P. putida KT2440 encode a second shikimate kinase isoenzyme (AroL/shikimate kinase II), and what is the relative contribution of AroK versus any paralog to in vivo shikimate kinase flux?
suggested_experiments:
- description: Determine steady-state kinetic parameters (Km/kcat for shikimate and ATP, Mg(2+) dependence) of recombinant KT2440 AroK to confirm substrate specificity and rule out promiscuous quinate kinase activity.
- description: Assess essentiality/fitness of an aroK deletion in KT2440 on minimal medium with and without aromatic amino acid supplementation to test for aromatic auxotrophy.
references:
- id: GO_REF:0000104
  title: Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features
  findings: []
- id: GO_REF:0000118
  title: TreeGrafter-generated GO annotations
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:12534463
  title: Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440.
  findings: []
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: KT2440 genome paper (Nelson et al. 2002); same PMID verified in aroA/aroB/aroC/aroE.

Gene Description

Existing Annotations Review

Core Functions

ATP- and Mg(2+)-dependent shikimate kinase that phosphorylates the 3-hydroxyl of shikimate to form shikimate 3-phosphate, catalyzing step 5 of the shikimate pathway in chorismate biosynthesis.

References

Suggested Questions for Experts

Suggested Experiments

Deep Research

Falcon

Research report: Functional annotation of aroK (UniProt Q88CV1, locus PP_5079) in Pseudomonas putida KT2440

0) Gene/protein identity verification (critical)

1) Key concepts and definitions (current understanding)

1.1 Shikimate pathway and the biochemical role of AroK

1.2 AroK versus AroL (bacterial isoenzymes)

1.3 Structure/function concepts relevant to substrate specificity

2) Functional annotation for P. putida KT2440 aroK (Q88CV1)

2.1 Molecular function (enzyme activity)

2.2 Biological process / pathway placement

2.3 Cellular localization (where it acts)

3) Recent developments and latest research (prioritizing 2023–2024)

3.1 KT2440 as an aromatics/synbio chassis (expert synthesis, 2024)

3.2 2024 “shikimate pathway-dependent catabolism” (SDC) rewiring to near-theoretical yields

3.3 2024 combinatorial pathway engineering to find bottlenecks (pABA)

4) Current applications and real-world implementations (with P. putida examples)

4.1 AroK as a practical flux-control lever in KT2440 muconate bioproduction

4.2 KT2440 production of gallic acid from glycerol (2023)

5) Expert opinions and analysis (authoritative sources)

5.1 Why KT2440 is repeatedly chosen for shikimate-pathway aromatics

6) Quantitative statistics and data points from recent and relevant studies

7) Evidence summary table

8) Practical functional-annotation statement (recommended)

9) Gaps and recommended next evidence to retrieve (to strengthen annotation)

Artifacts

Citations

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