GO Term	Evidence	Action	Reason
GO:0000287 magnesium ion binding	IEA GO_REF:0000104	KEEP AS NON CORE	Summary: Magnesium binding is a required cofactor feature but not the specific gene function. Reason: UniProt indicates two Mg(2+) ions per subunit for IlvC; this supports retention as non-core cofactor information. Supporting Evidence: file:PSEPK/ilvC/ilvC-uniprot.txt Binds 2 magnesium ions per subunit file:PSEPK/ilvC/ilvC-goa.tsv GO:0000287 magnesium ion binding file:PSEPK/ilvC/ilvC-deep-research-falcon.md IlvC requires NADPH for the reduction half-reaction and a divalent metal ion (most commonly Mg²⁺) to support the alkyl-migration/isomerization chemistry, which is mechanistically integrated in a single catalytic cycle for most KARIs.
GO:0004455 ketol-acid reductoisomerase activity	IEA GO_REF:0000120	ACCEPT	Summary: Ketol-acid reductoisomerase activity is the specific catalytic function of IlvC. Reason: The reviewed UniProt entry assigns EC 1.1.1.86 and describes the alkyl-migration/reduction reaction in BCAA biosynthesis. Supporting Evidence: file:PSEPK/ilvC/ilvC-uniprot.txt Catalyzes an alkyl-migration followed by a ketol-acid reduction file:PSEPK/ilvC/ilvC-goa.tsv GO:0004455 ketol-acid reductoisomerase activity file:PSEPK/ilvC/ilvC-deep-research-falcon.md Ketol-acid reductoisomerase (KARI; IlvC/AHAIR) is a conserved bacterial enzyme family within the 6-phosphogluconate dehydrogenase (6PGDH)-type superfamily that performs a chemically coupled alkyl-migration (isomerization) and NADP(H)-dependent reduction step in BCAA biosynthesis. file:PSEPK/ilvC/ilvC-deep-research-openscientist.md The gene _ilvC_ (ordered locus PP_4678; UniProt Q88DZ0*) of Pseudomonas putida* strain KT2440 (ATCC 47054 / DSM 6125 / NCIMB 11950) encodes ketol-acid reductoisomerase (KARI)
GO:0005829 cytosol	IEA GO_REF:0000118	KEEP AS NON CORE	Summary: Cytosol is plausible cellular context but not the defining function. Reason: The TreeGrafter cytosol annotation is consistent with a soluble bacterial biosynthetic enzyme but should remain non-core. Falcon deep research supports cytosolic localization by homology, noting no KT2440-specific localization experiment was retrieved. Supporting Evidence: file:PSEPK/ilvC/ilvC-goa.tsv GO:0005829 cytosol file:PSEPK/ilvC/ilvC-deep-research-falcon.md a Gram-negative bacterial IlvC homolog was reported as a soluble protein that could be purified without detergents, consistent with a cytosolic enzyme (as expected for a central-metabolism biosynthetic enzyme operating on cytosolic intermediates).
GO:0009082 branched-chain amino acid biosynthetic process	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: Branched-chain amino acid biosynthetic process is true but less specific than the valine/isoleucine branch terms. Reason: IlvC acts at a shared BCAA pathway step; the specific L-valine annotation and missing isoleucine annotation are more informative. Supporting Evidence: file:PSEPK/ilvC/ilvC-uniprot.txt Involved in the biosynthesis of branched-chain amino acids file:PSEPK/ilvC/ilvC-goa.tsv GO:0009082 branched-chain amino acid biosynthetic process file:PSEPK/ilvC/ilvC-deep-research-falcon.md IlvC is the second step in the pyruvate-to-2-ketoisovalerate segment of branched-chain amino-acid biosynthesis. It supports synthesis of valine and isoleucine directly and leucine indirectly via 2-oxoisovalerate-derived metabolism.
GO:0009099 L-valine biosynthetic process	IEA GO_REF:0000120	ACCEPT	Summary: IlvC participates directly in L-valine biosynthesis. Reason: UniProt maps IlvC to the L-valine biosynthesis route from pyruvate, step 2 of 4. Falcon deep research provides KT2440-specific genetic evidence (conditional essentiality on minimal medium and BCAA auxotrophy of ilvC mutants). Supporting Evidence: file:PSEPK/ilvC/ilvC-uniprot.txt L-valine from pyruvate: step 2/4 file:PSEPK/ilvC/ilvC-goa.tsv GO:0009099 L-valine biosynthetic process file:PSEPK/ilvC/ilvC-deep-research-falcon.md A genome-wide knockout screen on glucose minimal medium identified ilvC (PP4678) among genes whose disruption prevents growth on M9 minimal medium, i.e., conditionally essential in that environment.
GO:0016491 oxidoreductase activity	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: Oxidoreductase activity is a broad parent of the specific ketol-acid reductoisomerase function. Reason: The specific EC-linked ketol-acid reductoisomerase annotation should be preferred over the broad oxidoreductase parent. Supporting Evidence: file:PSEPK/ilvC/ilvC-uniprot.txt EC=1.1.1.86 file:PSEPK/ilvC/ilvC-goa.tsv GO:0016491 oxidoreductase activity
GO:0050661 NADP binding	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: NADP binding is a cofactor feature of IlvC. Reason: The reaction uses NADP/NADPH, but binding alone is less informative than the enzyme activity. Supporting Evidence: file:PSEPK/ilvC/ilvC-uniprot.txt NADP(+) file:PSEPK/ilvC/ilvC-goa.tsv GO:0050661 NADP binding file:PSEPK/ilvC/ilvC-deep-research-falcon.md IlvC requires NADPH for the reduction half-reaction and a divalent metal ion (most commonly Mg²⁺) to support the alkyl-migration/isomerization chemistry, which is mechanistically integrated in a single catalytic cycle for most KARIs.
GO:1901705 L-isoleucine biosynthetic process	IEA GO_REF:0000120	NEW	Summary: IlvC also participates directly in isoleucine biosynthesis. Reason: The reviewed UniProt entry has an L-isoleucine biosynthesis pathway statement, but the fetched GOA table lacks this specific process annotation. Falcon deep research independently reports isoleucine auxotrophy of KT2440 ilvC mutants, supporting a direct role in isoleucine biosynthesis. Supporting Evidence: file:PSEPK/ilvC/ilvC-uniprot.txt L-isoleucine from 2-oxobutanoate: step 2/4 file:PSEPK/ilvC/ilvC-deep-research-falcon.md ilvC mutants showed branched-chain amino-acid auxotrophy, with reported requirements involving isoleucine and valine/leucine supplementation depending on the assay context.

Deep Research

Falcon

(ilvC-deep-research-falcon.md)

this with annotations you find in gene/protein databases, but these can be outdated or inaccurate. Falcon Edison Scientific Literature 25 citations 2 artifacts 2026-06-02T17:08:46.170200

Edison artifact artifact-00 (text/markdown)

## Context ID: pqac-00000021 Figure 1 shows the engineered isobutanol biosynthetic pathway in Pseudomonas putida KT2440, which includes the genes alsS (acetolac (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 Pseudomonas putida KT2440 ilvC (UniProt Q88DZ0; locus PP_4678)

0) Target verification (critical disambiguation)

The requested target (UniProt Q88DZ0) corresponds to Pseudomonas putida KT2440 ilvC (locus PP_4678), which encodes ketol-acid reductoisomerase (KARI; also called acetohydroxyacid isomeroreductase, AHAIR; EC 1.1.1.86) and is part of the branched-chain amino-acid (BCAA) biosynthesis machinery. This identity is supported by KT2440 gene-essentiality screening explicitly listing PP4678 = ilvC and by multiple KT2440 metabolic-engineering studies that identify the native ilvCD locus and use it as the ketol-acid reductoisomerase (ilvC) and dihydroxyacid dehydratase (ilvD) module. (molina‐henares2010identificationofconditionally pages 2-3, nitschel2020engineeringpseudomonasputida pages 7-8)

1) Key concepts and current understanding

1.1 Definitions and enzyme class

Ketol-acid reductoisomerase (KARI; IlvC/AHAIR) is a conserved bacterial enzyme family within the 6-phosphogluconate dehydrogenase (6PGDH)-type superfamily that performs a chemically coupled alkyl-migration (isomerization) and NADP(H)-dependent reduction step in BCAA biosynthesis. (verdel‐aranda2015molecularannotationof pages 2-4)

1.2 Reaction and pathway position

In the canonical bacterial BCAA pathway, IlvC/AHAIR is the second enzyme in the pyruvate-to-2-ketoisovalerate segment (AHAS → AHAIR/IlvC → DHAD/IlvD). AHAS produces the IlvC substrates 2-acetolactate (valine/leucine branch) and 2-aceto-2-hydroxybutyrate (acetohydroxybutyrate) (isoleucine branch), and IlvC converts these toward the corresponding dihydroxy-acid intermediates for subsequent dehydration by IlvD. (lu2015characterizationandmodification pages 1-5)

A detailed mechanistic description from biochemical work indicates IlvC requires NADPH for the reduction half-reaction and a divalent metal ion (most commonly Mg²⁺) to support the alkyl-migration/isomerization chemistry, which is mechanistically integrated in a single catalytic cycle for most KARIs. (lu2015characterizationandmodification pages 5-9)

1.3 Substrate specificity and promiscuity

KARI substrate space is broader than a single physiological substrate pair; comparative enzyme studies emphasize that KARIs can show substrate promiscuity and that paralogs/orthologs can differ substantially in catalytic efficiencies across related keto/hydroxy-acid substrates relevant to valine and isoleucine precursor chemistry. (verdel‐aranda2015molecularannotationof pages 1-2, verdel‐aranda2015molecularannotationof pages 4-6)

1.4 Cofactor dependence (NADPH) and metal requirement

Primary biochemical assays for IlvC commonly use α-acetolactate as substrate and quantify activity by following NADPH oxidation at 340 nm, while including MgCl₂ in the reaction mixture. (lu2015characterizationandmodification pages 9-13)

A representative purified bacterial IlvC (from Ralstonia eutropha H16) shows Michaelis–Menten parameters: K_M(α-acetolactate) ≈ 6.2 mM, K_M(NADPH) ≈ 12.5 µM, and V_max ≈ 191 ± 3 mU/mg. (lu2015characterizationandmodification pages 34-39)

1.5 Cellular localization

No direct subcellular-localization experiment was retrieved for P. putida KT2440 IlvC specifically; however, a Gram-negative bacterial IlvC homolog was reported as a soluble protein that could be purified without detergents, consistent with a cytosolic enzyme (as expected for a central-metabolism biosynthetic enzyme operating on cytosolic intermediates). This provides supporting evidence for inferring cytosolic localization for KT2440 IlvC. (lu2015characterizationandmodification pages 13-17)

2) Pseudomonas putida KT2440-specific functional evidence

2.1 Conditional essentiality and auxotrophy phenotypes

A genome-wide knockout screen on glucose minimal medium identified ilvC (PP4678) among genes whose disruption prevents growth on M9 minimal medium, i.e., conditionally essential in that environment. (molina‐henares2010identificationofconditionally pages 2-3)

Phenotypic follow-up reported that ilvC (and ilvD) mutants show BCAA-related auxotrophies: ilvC/ilvD mutants were described as requiring valine and leucine for growth in one context and being classified under isoleucine auxotrophs with partial rescue by L-isoleucine in another table-based summary, indicating that loss of IlvC disrupts multiple branches of BCAA supply under minimal conditions. (molina‐henares2010identificationofconditionally pages 7-9, molina‐henares2010identificationofconditionally pages 6-7)

2.2 Integration in KT2440 redox and carbon-flux engineering

Because IlvC is NADPH-dependent, its activity becomes a key constraint when KT2440 is engineered to overproduce 2-ketoisovalerate (2-KIV) or isobutanol. In engineered KT2440 isobutanol pathways, IlvC functions alongside AlsS (AHAS substitute) and IlvD to generate 2-KIV, which is then decarboxylated and reduced to isobutanol; the pathway is explicitly described as NADPH-consuming, and KT2440 engineering includes redox-balancing steps such as deleting soluble transhydrogenase sthA. (nitschel2020engineeringpseudomonasputida pages 8-10, nitschel2020engineeringpseudomonasputida pages 7-8)

3) Recent developments (prioritizing 2023–2024)

3.1 2023: Tunable growth/product “metabolic valve” in P. putida KT2440

A 2023 Metabolic Engineering study implemented a tunable PDH (pyruvate dehydrogenase) valve to control growth while enabling overflow of pyruvate, and coupled this with a 2-KIV module containing alsS-ilvC-ilvD (native ilvC/ilvD plus heterologous alsS) under inducible control. This work highlights ilvC as a core lever for converting pyruvate into BCAA-derived platform intermediates in KT2440. (batianis2023atunablemetabolic pages 4-5)

3.2 2024: Authoritative review synthesis for isobutanol pathways

A 2024 review (Applied Microbiology and Biotechnology) positions ilvC/KARI (AHAIR) as the second enzymatic step converting 2-acetolactate to 2,3-dihydroxyisovalerate in BCAA-derived isobutanol pathways and emphasizes that redox/cofactor engineering is a recurring requirement for improving isobutanol production (cofactor deficiency/redox imbalance is widely treated as a bottleneck), alongside pathway overexpression and blocking competing routes. (Published Jan 2024; https://doi.org/10.1007/s00253-023-12821-9) (nawab2024microbialhostengineering pages 1-3, nawab2024microbialhostengineering pages 6-8)

3.3 2024: Cofactor rewiring of IlvC to shift NADPH → NADH usage

A 2024 Microbial Cell Factories study demonstrates modern cofactor-engineering directly targeting ilvC/AHAIR: a triple mutant L67E/R68F/K75E (AHAIRM) was described as shifting preference from NADPH toward NADH, and increasing expression of this NADH-utilizing AHAIR increased carbon flux to α-ketoisovalerate (including an increase of diverted carbon ratio from 32.9% to 48.4%) and enabled high KIV titers (e.g., 18.8 g/L from 60 g/L glucose in 21 h; and up to ~40.7 g/L from whey powder in fed-batch with yield ~0.418 g/g lactose). Although this is not in Pseudomonas, it is directly relevant to interpreting ilvC/KARI as a redox bottleneck and a design target. (Published Oct 2024; https://doi.org/10.1186/s12934-024-02545-4) (sun2024productionofαketoisovalerate pages 6-8, sun2024productionofαketoisovalerate pages 8-9)

4) Current applications and real-world implementations

4.1 Industrial biotechnology: BCAA-pathway-derived chemicals in P. putida

In KT2440, ilvC is widely used as part of engineered modules for producing isobutanol, a next-generation biofuel/solvent. A representative KT2440 study overexpressed native ilvC/ilvD and incorporated heterologous steps to convert 2-KIV to isobutanol, achieving 22 ± 2 mg isobutanol per g glucose under aerobic conditions. (Published Feb 2020; https://doi.org/10.1002/elsc.201900151) (nitschel2020engineeringpseudomonasputida pages 1-2)

A subsequent KT2440 bioprocess study scaled production and showed that shifting to micro-aerobic production conditions can improve conversion yield and reduce undesired carbon loss, reporting 3.35 g/L isobutanol in a two-stage process and an integral yield of 60 mg/g glucose under micro-aerobic production conditions. (Published Mar 2021; https://doi.org/10.1002/elsc.202000116) (ankenbauer2021micro‐aerobicproductionof pages 1-2, ankenbauer2021micro‐aerobicproductionof pages 8-10)

4.2 Systems metabolic engineering: growth-coupled production control (2023)

The 2023 “metabolic valve” approach explicitly treats the ilvC-containing 2-KIV module as a controllable sink for pyruvate, enabling engineered coupling between growth control (via PDH modulation) and production potential for BCAA-derived intermediates. (Published Jan 2023; https://doi.org/10.1016/j.ymben.2022.10.002) (batianis2023atunablemetabolic pages 4-5)

5) Expert opinions / analysis (authoritative synthesis)

5.1 IlvC as a predictable redox bottleneck

Across engineered BCAA-derived alcohol/ketoacid pathways, IlvC/KARI is repeatedly implicated as a redox-limited node because it uses NADPH, while many central metabolic processes regenerate NADH more readily. The 2024 review frames “cofactor engineering” as a standard strategy to overcome these limitations alongside enzyme overexpression and competing-pathway deletions. (nawab2024microbialhostengineering pages 1-3, nawab2024microbialhostengineering pages 6-8)

5.2 Practical interpretation for KT2440 functional annotation

For annotation purposes, the strongest KT2440-specific evidence supports: (i) IlvC is essential for de novo BCAA synthesis in minimal conditions (auxotrophy/conditional essentiality), and (ii) IlvC is a high-leverage enzyme for redirecting carbon from pyruvate into 2-KIV/isobutanol modules, with system-level consequences driven by NADPH availability and transhydrogenase activity. (molina‐henares2010identificationofconditionally pages 2-3, nitschel2020engineeringpseudomonasputida pages 7-8)

6) Key quantitative statistics and data points

The table below consolidates the most directly supported functional-annotation and application data for KT2440 ilvC and closely related IlvC biochemistry.

Aspect	Key findings	Evidence type	Best citations
Identity	PP_4678 in Pseudomonas putida KT2440 is annotated as ilvC, encoding ketol-acid reductoisomerase/acetohydroxyacid isomeroreductase (KARI/AHAIR; EC 1.1.1.86). Independent P. putida engineering studies place native ilvC in the ilvCD locus used for branched-chain ketoacid formation, matching UniProt Q88DZ0.	Experimental, engineering, inference	(nitschel2020engineeringpseudomonasputida pages 7-8, molina‐henares2010identificationofconditionally pages 2-3)
Reaction	IlvC/KARI catalyzes the coupled isomerization plus reduction step of branched-chain amino-acid biosynthesis, converting AHAS products toward dihydroxy-acid intermediates on the route to 2-ketoisovalerate and related branched-chain precursors.	Experimental, inference	(verdel‐aranda2015molecularannotationof pages 2-4, lu2015characterizationandmodification pages 1-5)
Substrates/cofactors	Physiological substrates include acetolactate/acetohydroxybutyrate pathway intermediates; assays for bacterial IlvC use α-acetolactate as substrate and monitor NADPH oxidation. KARI generally requires a divalent metal ion, usually Mg²⁺, for the alkyl-migration step.	Experimental, inference	(lu2015characterizationandmodification pages 9-13, lu2015characterizationandmodification pages 5-9)
Pathway role	IlvC is the second step in the pyruvate-to-2-ketoisovalerate segment of branched-chain amino-acid biosynthesis. It supports synthesis of valine and isoleucine directly and leucine indirectly via 2-oxoisovalerate-derived metabolism.	Experimental, inference	(lu2015characterizationandmodification pages 1-5, molina‐henares2010identificationofconditionally pages 7-9)
Localization	No direct localization study was found for P. putida IlvC, but bacterial IlvC/AHAIR is characterized as a soluble/cytosolic enzyme in related Gram-negative bacteria, with purification from soluble lysate and no detergent requirement; this supports a cytosolic localization inference for KT2440 IlvC.	Experimental in homolog, inference for P. putida	(lu2015characterizationandmodification pages 13-17, lu2015characterizationandmodification pages 9-13)
Genetics/phenotype in P. putida	In a genome-wide mutant screen, ilvC (PP4678) was identified as conditionally essential for growth on glucose minimal medium. ilvC mutants showed branched-chain amino-acid auxotrophy, with reported requirements involving isoleucine and valine/leucine supplementation depending on the assay context.	Experimental genetics	(molina‐henares2010identificationofconditionally pages 2-3, molina‐henares2010identificationofconditionally pages 7-9, molina‐henares2010identificationofconditionally pages 6-7)
Engineering applications	Native P. putida ilvC has been repeatedly overexpressed with ilvD (and often alsS) to channel pyruvate into 2-ketoisovalerate and isobutanol production. Recent KT2440 work also embeds ilvC in tunable metabolic-valve designs that connect growth control with ketoacid overproduction.	Engineering	(nitschel2020engineeringpseudomonasputida pages 1-2, batianis2023atunablemetabolic pages 6-7, batianis2023atunablemetabolic pages 4-5)
Quantitative data	Engineered P. putida KT2440 strains overexpressing native ilvC/ilvD produced isobutanol at 22 ± 2 mg/g glucose aerobically, while microaerobic processing improved glucose-to-isobutanol yield to 60 mg/g glucose and a 30 L process reached 3.35 g/L. For a characterized bacterial IlvC homolog, reported kinetics include K_M ~6.2 mM for α-acetolactate, K_M 12.5 µM for NADPH, and V_max 191 ± 3 mU/mg.	Engineering, experimental biochemistry	(nitschel2020engineeringpseudomonasputida pages 1-2, lu2015characterizationandmodification pages 34-39, lu2015characterizationandmodification pages 13-17)

Table: This table summarizes the main functional annotation evidence for Pseudomonas putida KT2440 ilvC (Q88DZ0/PP_4678), including biochemical role, pathway context, localization inference, genetics, and engineering relevance. It is useful as a compact evidence map linking species-specific findings to broader KARI knowledge.

Additional visual support: Nitschel et al. include a pathway schematic showing the engineered module alsS–ilvC–ilvD and a strain table listing Y_Iso/S values (mg/g glucose) for KT2440 engineered strains. (nitschel2020engineeringpseudomonasputida media 681e76b1, nitschel2020engineeringpseudomonasputida media 2f7b9801)

7) Consolidated functional annotation (recommended)

Gene: ilvC (PP_4678) in P. putida KT2440.

Protein function (primary): Cytosolic ketol-acid reductoisomerase/acetohydroxyacid isomeroreductase (KARI/AHAIR; EC 1.1.1.86) catalyzing the NADPH-dependent isomerization+reduction of acetohydroxy-acid intermediates (from AHAS/AlsS) to dihydroxy-acid products in the BCAA pathway. (verdel‐aranda2015molecularannotationof pages 2-4, lu2015characterizationandmodification pages 5-9)

Substrate specificity (physiological): Acts on intermediates arising from 2-acetolactate and acetohydroxybutyrate branches that feed valine/isoleucine (and indirectly leucine) biosynthesis; KARI family members can show promiscuity among related keto/hydroxy-acid substrates. (lu2015characterizationandmodification pages 1-5, verdel‐aranda2015molecularannotationof pages 1-2)

Cofactors/requirements: NADPH is the primary hydride donor; many KARIs require Mg²⁺ (or another divalent metal) for the isomerization step. (lu2015characterizationandmodification pages 5-9, lu2015characterizationandmodification pages 9-13)

Biological process/pathway: Branched-chain amino-acid biosynthesis; supplies precursors for valine/isoleucine (and leucine via downstream steps). (molina‐henares2010identificationofconditionally pages 7-9, lu2015characterizationandmodification pages 1-5)

Cellular localization: No KT2440-specific localization experiment was retrieved, but homolog evidence supports IlvC as a soluble/cytosolic enzyme; consistent with its function in cytosolic amino-acid biosynthesis. (lu2015characterizationandmodification pages 13-17)

Genetic evidence in KT2440: ilvC is conditionally essential for growth on glucose minimal medium and its disruption yields BCAA auxotrophy (reported requirements include isoleucine and valine/leucine supplementation). (molina‐henares2010identificationofconditionally pages 2-3, molina‐henares2010identificationofconditionally pages 7-9, molina‐henares2010identificationofconditionally pages 6-7)

8) Limitations of this evidence set

A direct, P. putida KT2440-specific purified-enzyme kinetic characterization and an in vivo localization assay for Q88DZ0 were not retrieved in the available corpus; thus, enzyme kinetics and localization are supported by KT2440 pathway-engineering/phenotype data and by biochemical/localization data from closely related bacteria and IlvC homologs rather than by a KT2440-specific biochemical study. (lu2015characterizationandmodification pages 13-17, lu2015characterizationandmodification pages 34-39)

References

(molina‐henares2010identificationofconditionally pages 2-3): M. Antonia Molina‐Henares, Jesús De La Torre, Adela García‐Salamanca, A. Jesús Molina‐Henares, M. Carmen Herrera, Juan L. Ramos, and Estrella Duque. Identification of conditionally essential genes for growth of pseudomonas putida kt2440 on minimal medium through the screening of a genome‐wide mutant library. Environmental Microbiology, 12:1468-1485, Jun 2010. URL: https://doi.org/10.1111/j.1462-2920.2010.02166.x, doi:10.1111/j.1462-2920.2010.02166.x. This article has 89 citations and is from a domain leading peer-reviewed journal.
(nitschel2020engineeringpseudomonasputida pages 7-8): Robert Nitschel, Andreas Ankenbauer, Ilona Welsch, Nicolas T. Wirth, Christoph Massner, Naveed Ahmad, Stephen McColm, Frédéric Borges, Ian Fotheringham, Ralf Takors, and Bastian Blombach. Engineering pseudomonas putida kt2440 for the production of isobutanol. Engineering in Life Sciences, 20:148-159, Feb 2020. URL: https://doi.org/10.1002/elsc.201900151, doi:10.1002/elsc.201900151. This article has 32 citations and is from a peer-reviewed journal.
(verdel‐aranda2015molecularannotationof pages 2-4): Karina Verdel‐Aranda, Susana T. López‐Cortina, David A. Hodgson, and Francisco Barona‐Gómez. Molecular annotation of ketol-acid reductoisomerases from streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity. Microbial Biotechnology, 8:239-252, Oct 2015. URL: https://doi.org/10.1111/1751-7915.12175, doi:10.1111/1751-7915.12175. This article has 22 citations and is from a peer-reviewed journal.
(lu2015characterizationandmodification pages 1-5): Jingnan Lu, Christopher J. Brigham, Jens K. Plassmeier, and Anthony J. Sinskey. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of ralstonia eutropha h16. Applied Microbiology and Biotechnology, 99:761-774, Aug 2015. URL: https://doi.org/10.1007/s00253-014-5965-3, doi:10.1007/s00253-014-5965-3. This article has 21 citations and is from a domain leading peer-reviewed journal.
(lu2015characterizationandmodification pages 5-9): Jingnan Lu, Christopher J. Brigham, Jens K. Plassmeier, and Anthony J. Sinskey. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of ralstonia eutropha h16. Applied Microbiology and Biotechnology, 99:761-774, Aug 2015. URL: https://doi.org/10.1007/s00253-014-5965-3, doi:10.1007/s00253-014-5965-3. This article has 21 citations and is from a domain leading peer-reviewed journal.
(verdel‐aranda2015molecularannotationof pages 1-2): Karina Verdel‐Aranda, Susana T. López‐Cortina, David A. Hodgson, and Francisco Barona‐Gómez. Molecular annotation of ketol-acid reductoisomerases from streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity. Microbial Biotechnology, 8:239-252, Oct 2015. URL: https://doi.org/10.1111/1751-7915.12175, doi:10.1111/1751-7915.12175. This article has 22 citations and is from a peer-reviewed journal.
(verdel‐aranda2015molecularannotationof pages 4-6): Karina Verdel‐Aranda, Susana T. López‐Cortina, David A. Hodgson, and Francisco Barona‐Gómez. Molecular annotation of ketol-acid reductoisomerases from streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity. Microbial Biotechnology, 8:239-252, Oct 2015. URL: https://doi.org/10.1111/1751-7915.12175, doi:10.1111/1751-7915.12175. This article has 22 citations and is from a peer-reviewed journal.
(lu2015characterizationandmodification pages 9-13): Jingnan Lu, Christopher J. Brigham, Jens K. Plassmeier, and Anthony J. Sinskey. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of ralstonia eutropha h16. Applied Microbiology and Biotechnology, 99:761-774, Aug 2015. URL: https://doi.org/10.1007/s00253-014-5965-3, doi:10.1007/s00253-014-5965-3. This article has 21 citations and is from a domain leading peer-reviewed journal.
(lu2015characterizationandmodification pages 34-39): Jingnan Lu, Christopher J. Brigham, Jens K. Plassmeier, and Anthony J. Sinskey. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of ralstonia eutropha h16. Applied Microbiology and Biotechnology, 99:761-774, Aug 2015. URL: https://doi.org/10.1007/s00253-014-5965-3, doi:10.1007/s00253-014-5965-3. This article has 21 citations and is from a domain leading peer-reviewed journal.
(lu2015characterizationandmodification pages 13-17): Jingnan Lu, Christopher J. Brigham, Jens K. Plassmeier, and Anthony J. Sinskey. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of ralstonia eutropha h16. Applied Microbiology and Biotechnology, 99:761-774, Aug 2015. URL: https://doi.org/10.1007/s00253-014-5965-3, doi:10.1007/s00253-014-5965-3. This article has 21 citations and is from a domain leading peer-reviewed journal.
(molina‐henares2010identificationofconditionally pages 7-9): M. Antonia Molina‐Henares, Jesús De La Torre, Adela García‐Salamanca, A. Jesús Molina‐Henares, M. Carmen Herrera, Juan L. Ramos, and Estrella Duque. Identification of conditionally essential genes for growth of pseudomonas putida kt2440 on minimal medium through the screening of a genome‐wide mutant library. Environmental Microbiology, 12:1468-1485, Jun 2010. URL: https://doi.org/10.1111/j.1462-2920.2010.02166.x, doi:10.1111/j.1462-2920.2010.02166.x. This article has 89 citations and is from a domain leading peer-reviewed journal.
(molina‐henares2010identificationofconditionally pages 6-7): M. Antonia Molina‐Henares, Jesús De La Torre, Adela García‐Salamanca, A. Jesús Molina‐Henares, M. Carmen Herrera, Juan L. Ramos, and Estrella Duque. Identification of conditionally essential genes for growth of pseudomonas putida kt2440 on minimal medium through the screening of a genome‐wide mutant library. Environmental Microbiology, 12:1468-1485, Jun 2010. URL: https://doi.org/10.1111/j.1462-2920.2010.02166.x, doi:10.1111/j.1462-2920.2010.02166.x. This article has 89 citations and is from a domain leading peer-reviewed journal.
(nitschel2020engineeringpseudomonasputida pages 8-10): Robert Nitschel, Andreas Ankenbauer, Ilona Welsch, Nicolas T. Wirth, Christoph Massner, Naveed Ahmad, Stephen McColm, Frédéric Borges, Ian Fotheringham, Ralf Takors, and Bastian Blombach. Engineering pseudomonas putida kt2440 for the production of isobutanol. Engineering in Life Sciences, 20:148-159, Feb 2020. URL: https://doi.org/10.1002/elsc.201900151, doi:10.1002/elsc.201900151. This article has 32 citations and is from a peer-reviewed journal.
(batianis2023atunablemetabolic pages 4-5): Christos Batianis, Rik P. van Rosmalen, Monika Major, Cheyenne van Ee, Alexandros Kasiotakis, Ruud A. Weusthuis, and Vitor A.P. Martins dos Santos. A tunable metabolic valve for precise growth control and increased product formation in pseudomonas putida. Metabolic Engineering, 75:47-57, Jan 2023. URL: https://doi.org/10.1016/j.ymben.2022.10.002, doi:10.1016/j.ymben.2022.10.002. This article has 19 citations and is from a domain leading peer-reviewed journal.
(nawab2024microbialhostengineering pages 1-3): Said Nawab, YaFei Zhang, Muhammad Wajid Ullah, Adil Farooq Lodhi, Syed Bilal Shah, Mujeeb Ur Rahman, and Yang-Chun Yong. Microbial host engineering for sustainable isobutanol production from renewable resources. Applied Microbiology and Biotechnology, 108:1-18, Jan 2024. URL: https://doi.org/10.1007/s00253-023-12821-9, doi:10.1007/s00253-023-12821-9. This article has 12 citations and is from a domain leading peer-reviewed journal.
(nawab2024microbialhostengineering pages 6-8): Said Nawab, YaFei Zhang, Muhammad Wajid Ullah, Adil Farooq Lodhi, Syed Bilal Shah, Mujeeb Ur Rahman, and Yang-Chun Yong. Microbial host engineering for sustainable isobutanol production from renewable resources. Applied Microbiology and Biotechnology, 108:1-18, Jan 2024. URL: https://doi.org/10.1007/s00253-023-12821-9, doi:10.1007/s00253-023-12821-9. This article has 12 citations and is from a domain leading peer-reviewed journal.
(sun2024productionofαketoisovalerate pages 6-8): Weikang Sun, Shuo Wang, Xiaoxu Tan, Leilei Guo, Wei Liu, Wenjia Tian, Hui Zhang, Tianyi Jiang, Wensi Meng, Yidong Liu, Zhaoqi Kang, Chuanjuan Lü, Chao Gao, Ping Xu, and Cuiqing Ma. Production of α-ketoisovalerate with whey powder by systemic metabolic engineering of klebsiella oxytoca. Microbial Cell Factories, Oct 2024. URL: https://doi.org/10.1186/s12934-024-02545-4, doi:10.1186/s12934-024-02545-4. This article has 2 citations and is from a peer-reviewed journal.
(sun2024productionofαketoisovalerate pages 8-9): Weikang Sun, Shuo Wang, Xiaoxu Tan, Leilei Guo, Wei Liu, Wenjia Tian, Hui Zhang, Tianyi Jiang, Wensi Meng, Yidong Liu, Zhaoqi Kang, Chuanjuan Lü, Chao Gao, Ping Xu, and Cuiqing Ma. Production of α-ketoisovalerate with whey powder by systemic metabolic engineering of klebsiella oxytoca. Microbial Cell Factories, Oct 2024. URL: https://doi.org/10.1186/s12934-024-02545-4, doi:10.1186/s12934-024-02545-4. This article has 2 citations and is from a peer-reviewed journal.
(nitschel2020engineeringpseudomonasputida pages 1-2): Robert Nitschel, Andreas Ankenbauer, Ilona Welsch, Nicolas T. Wirth, Christoph Massner, Naveed Ahmad, Stephen McColm, Frédéric Borges, Ian Fotheringham, Ralf Takors, and Bastian Blombach. Engineering pseudomonas putida kt2440 for the production of isobutanol. Engineering in Life Sciences, 20:148-159, Feb 2020. URL: https://doi.org/10.1002/elsc.201900151, doi:10.1002/elsc.201900151. This article has 32 citations and is from a peer-reviewed journal.
(ankenbauer2021micro‐aerobicproductionof pages 1-2): Andreas Ankenbauer, Robert Nitschel, Attila Teleki, Tobias Müller, Lorenzo Favilli, Bastian Blombach, and Ralf Takors. Micro‐aerobic production of isobutanol with engineered pseudomonas putida. Engineering in Life Sciences, 21:475-488, Mar 2021. URL: https://doi.org/10.1002/elsc.202000116, doi:10.1002/elsc.202000116. This article has 19 citations and is from a peer-reviewed journal.
(ankenbauer2021micro‐aerobicproductionof pages 8-10): Andreas Ankenbauer, Robert Nitschel, Attila Teleki, Tobias Müller, Lorenzo Favilli, Bastian Blombach, and Ralf Takors. Micro‐aerobic production of isobutanol with engineered pseudomonas putida. Engineering in Life Sciences, 21:475-488, Mar 2021. URL: https://doi.org/10.1002/elsc.202000116, doi:10.1002/elsc.202000116. This article has 19 citations and is from a peer-reviewed journal.
(batianis2023atunablemetabolic pages 6-7): Christos Batianis, Rik P. van Rosmalen, Monika Major, Cheyenne van Ee, Alexandros Kasiotakis, Ruud A. Weusthuis, and Vitor A.P. Martins dos Santos. A tunable metabolic valve for precise growth control and increased product formation in pseudomonas putida. Metabolic Engineering, 75:47-57, Jan 2023. URL: https://doi.org/10.1016/j.ymben.2022.10.002, doi:10.1016/j.ymben.2022.10.002. This article has 19 citations and is from a domain leading peer-reviewed journal.
(nitschel2020engineeringpseudomonasputida media 681e76b1): Robert Nitschel, Andreas Ankenbauer, Ilona Welsch, Nicolas T. Wirth, Christoph Massner, Naveed Ahmad, Stephen McColm, Frédéric Borges, Ian Fotheringham, Ralf Takors, and Bastian Blombach. Engineering pseudomonas putida kt2440 for the production of isobutanol. Engineering in Life Sciences, 20:148-159, Feb 2020. URL: https://doi.org/10.1002/elsc.201900151, doi:10.1002/elsc.201900151. This article has 32 citations and is from a peer-reviewed journal.
(nitschel2020engineeringpseudomonasputida media 2f7b9801): Robert Nitschel, Andreas Ankenbauer, Ilona Welsch, Nicolas T. Wirth, Christoph Massner, Naveed Ahmad, Stephen McColm, Frédéric Borges, Ian Fotheringham, Ralf Takors, and Bastian Blombach. Engineering pseudomonas putida kt2440 for the production of isobutanol. Engineering in Life Sciences, 20:148-159, Feb 2020. URL: https://doi.org/10.1002/elsc.201900151, doi:10.1002/elsc.201900151. This article has 32 citations and is from a peer-reviewed journal.

Artifacts

Edison artifact artifact-00

Citations

lu2015characterizationandmodification pages 1-5
lu2015characterizationandmodification pages 5-9
lu2015characterizationandmodification pages 9-13
lu2015characterizationandmodification pages 34-39
lu2015characterizationandmodification pages 13-17
batianis2023atunablemetabolic pages 4-5
nitschel2020engineeringpseudomonasputida pages 1-2
nitschel2020engineeringpseudomonasputida pages 7-8
nitschel2020engineeringpseudomonasputida pages 8-10
nawab2024microbialhostengineering pages 1-3
nawab2024microbialhostengineering pages 6-8
batianis2023atunablemetabolic pages 6-7
https://doi.org/10.1007/s00253-023-12821-9
https://doi.org/10.1186/s12934-024-02545-4
https://doi.org/10.1002/elsc.201900151
https://doi.org/10.1002/elsc.202000116
https://doi.org/10.1016/j.ymben.2022.10.002
https://doi.org/10.1111/j.1462-2920.2010.02166.x,
https://doi.org/10.1002/elsc.201900151,
https://doi.org/10.1111/1751-7915.12175,
https://doi.org/10.1007/s00253-014-5965-3,
https://doi.org/10.1016/j.ymben.2022.10.002,
https://doi.org/10.1007/s00253-023-12821-9,
https://doi.org/10.1186/s12934-024-02545-4,
https://doi.org/10.1002/elsc.202000116,

OpenScientist

(ilvC-deep-research-openscientist.md)

Functional Annotation Report: *ilvC* / Ketol-Acid Reductoisomerase (Q88DZ0) in *Pseudomonas putida* KT2440 OpenScientist openscientist-autonomous 6 citations 2 artifacts 2026-07-17T13:50:37.186556

OpenScientist final report (text/html)

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Functional Annotation Report: ilvC / Ketol-Acid Reductoisomerase (Q88DZ0) in Pseudomonas putida KT2440

Summary

The gene ilvC (ordered locus PP_4678; UniProt Q88DZ0) of Pseudomonas putida strain KT2440 (ATCC 47054 / DSM 6125 / NCIMB 11950) encodes ketol-acid reductoisomerase (KARI), also called acetohydroxy-acid isomeroreductase (AHIR/AHAIR), EC 1.1.1.86. This is the second enzyme of the branched-chain amino acid (BCAA) biosynthesis pathway, which produces L-valine, L-isoleucine, and L-leucine. The enzyme is a soluble, cytoplasmic, 338-residue class I ("type 1") KARI that uses two Mg²⁺ ions and NADPH to carry out, within a single active site, two chemically distinct reactions: an Mg²⁺-dependent alkyl (methyl or ethyl) migration/isomerization followed by an NADPH-dependent reduction. Gene identity was rigorously verified: the gene symbol, organism, EC number, protein family (ketol-acid reductoisomerase family), and the diagnostic InterPro domains (KARI_N IPR013116, KARI_C IPR000506, KARI_prok IPR014359) are all internally consistent, and the recovered literature describes exactly this enzyme class. There is no ambiguity — ilvC unambiguously denotes KARI.

Mechanistically, KARI converts (S)-2-acetolactate → (2R)-2,3-dihydroxy-3-isovalerate (the valine/leucine branch) and (S)-2-aceto-2-hydroxybutyrate → (2R,3R)-2,3-dihydroxy-3-methylvalerate (the isoleucine branch). It sits immediately downstream of acetohydroxyacid synthase (AHAS, encoded by ilvBN, which supplies its substrates) and immediately upstream of dihydroxyacid dehydratase (DHAD, ilvD). In P. putida KT2440, PP_4678 (ilvC) is genomically clustered with AHAS genes in a canonical ilvBN-ilvC-type arrangement, placing the enzyme physically and transcriptionally adjacent to its substrate-producing partner. The product ultimately feeds not only the three BCAAs but also 2-ketoisovalerate-derived pantothenate and coenzyme A biosynthesis.

The enzyme carries out its function in the cytoplasm, where the entire soluble BCAA biosynthetic pathway resides. Because this pathway is present in plants, fungi, and bacteria but absent in animals, KARI is a validated target for antimicrobials and herbicides, and — because of its NADPH dependence — a heavily engineered node in microbial isobutanol biofuel production. The findings below are supported by four converging lines of evidence: (1) authoritative enzymological and structural literature on KARI mechanism, (2) the curated UniProt/HAMAP annotation specific to Q88DZ0, (3) genomic-context analysis of the PP_4678 locus in KT2440, and (4) direct bioinformatic analysis of the Q88DZ0 sequence identifying its cofactor-binding and metal-coordinating residues.

Key Findings

F001 — ilvC encodes ketol-acid reductoisomerase, the second step of BCAA biosynthesis

The core functional assignment is definitive. Q88DZ0 is annotated as EC 1.1.1.86 under HAMAP-Rule MF_00435. KARI is a bifunctional enzyme that catalyzes two chemically distinct steps within a single active site: (1) an Mg²⁺-dependent alkyl migration (isomerization), in which a methyl or ethyl group migrates to convert (S)-2-acetolactate to 3-hydroxy-3-methyl-2-oxobutanoate, and (2) an NADPH-dependent reduction of the resulting keto group. The net conversions are:

Valine/leucine branch: (S)-2-acetolactate → (2R)-2,3-dihydroxy-3-isovalerate
Isoleucine branch: 2-aceto-2-hydroxybutyrate → (2R,3R)-2,3-dihydroxy-3-methylvalerate

The substrate specificity is strict for the acetohydroxy-acid intermediates of the ilv pathway. This is directly established by primary enzymology: KARI "catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions—alkyl migration and reduction—and requires Mg(2+) and NADPH for activity" (PMID: 19362563). Its ordinal position is equally clear: "Ketol-acid reductoisomerase (KARI) is the second enzyme in the branched-chain amino acid biosynthesis pathway" (PMID: 23036858).

F002 — KARI is an Mg²⁺/NADPH-dependent, two-domain enzyme using an induced-fit mechanism

Structurally, KARI is built from two domains: an N-terminal Rossmann-like NAD(P)H-binding domain (KARI_N, IPR013116) and a C-terminal knotted α-helical domain (KARI_C, IPR000506) that houses the two catalytic Mg²⁺ ions. The active site lies at the interface between the two domains. Crystallographic studies demonstrate that the binding of Mg²⁺ and NADPH triggers a domain-closure/reorganization (induced fit) that assembles the catalytically competent active site. As stated for the plant enzyme, "the binding of Mg(2+) and NADPH opens the interface between the N- and C-domains, thereby allowing access for the substrates to bind" (PMID: 23036858). For the class I enzymes to which the prokaryotic ilvC belongs, "the class I KARI structures indicate that the active sites close upon binding NAD(P)H" (PMID: 25849365), directly confirming the induced-fit closure mechanism. Isothermal titration calorimetry on the E. coli enzyme further shows Mg²⁺ binding increases conformational disorder while NADPH binding increases rigidity, consistent with the ordered assembly of the active site.

F003 — Q88DZ0 is a class I / "type 1" prokaryotic KARI

UniProt annotates Q88DZ0 explicitly as "Ketol-acid reductoisomerase type 1 / type I" and assigns the prokaryotic KARI signature (KARI_prok, IPR014359). KARIs partition into two classes: class I (short, single ~340-residue subunit, predominant in bacteria and fungi) and class II (arising from an internal duplication of the C-domain, ~490+ residues, found in plants and some bacteria such as E. coli). Q88DZ0's 338-residue length and prokaryotic signature place it firmly in class I. The literature notes that "most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, [while] structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication" (PMID: 25849365). The class distinction is functionally relevant because it correlates with the length of the β2αB cofactor-specificity loop that governs NADPH-versus-NADH preference.

F004 — KARI operates in the cytoplasm; it is a validated antimicrobial target absent from animals and a key biofuel-engineering node

The BCAA biosynthesis pathway — and KARI with it — is "found in plants, fungi and bacteria but not in animals. This difference in metabolism between animals and microorganisms makes KARI an attractive target for the development of antimicrobial agents" (PMID: 23036858). The functional importance of KARI in vivo is demonstrated genetically across taxa: in Mycobacterium tuberculosis, "Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation" (PMID: 36354071); and in the fungal pathogen Fusarium graminearum, deletion of the KARI homologue FgIlv5 abolishes growth without exogenous isoleucine and valine and reduces virulence (PMID: 24493249). As a soluble cytosolic enzyme, KARI is also a central node in engineered isobutanol production. Because glycolysis yields NADH while wild-type KARI uses NADPH, cofactor-swapped variants were engineered: "an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield" (PMID: 21515217), underscoring KARI's NADPH specificity as the natural default and its role as an engineerable bottleneck.

F005 — Protein-specific annotation: 338-residue two-domain enzyme, 2 Mg²⁺/subunit, step 2/4 of both Val and Ile routes

The curated UniProt Q88DZ0 record (HAMAP-Rule MF_00435) provides protein-specific detail. The sequence is 338 amino acids long, consistent with a compact single-subunit class I KARI. Its domain architecture comprises an N-terminal KARI Rossmann domain (~residues 1–181) containing the Rossmann NAD(P)-binding fingerprint (…GYGSQGHA… around residues 22–29) and a C-terminal KARI knotted domain (~182–327). An active-site residue is annotated at position 107, with multiple metal/substrate binding sites (residues 24–27, 47, 50, 52, 82–85, 133, 190, 194, 226, 230, 251). The enzyme binds two Mg²⁺ per subunit and uses NADPH. The curated Rhea reactions are:

RHEA:22068 — (2R)-2,3-dihydroxy-3-methylbutanoate + NADP⁺ = (2S)-2-acetolactate + NADPH + H⁺
RHEA:13493 — (2R,3R)-2,3-dihydroxy-3-methylpentanoate + NADP⁺ = (S)-2-ethyl-2-hydroxy-3-oxobutanoate + NADPH + H⁺

Pathway assignments are L-valine biosynthesis from pyruvate, step 2/4 and L-isoleucine biosynthesis from 2-oxobutanoate, step 2/4.

F006 — PP_4678 is genomically clustered with acetohydroxyacid synthase genes and feeds Val/Ile and pantothenate/CoA pathways

Genomic-context analysis of P. putida KT2440 (KEGG/GenBank) places PP_4678 (ilvC) at complement(5316083..5317099), immediately preceded on the same minus strand by PP_4679 (~163 aa, acetohydroxyacid synthase small/regulatory subunit, ilvN/ilvH-like; 57-bp gap to ilvC) and PP_4680 (~574 aa, acetohydroxyacid synthase large/catalytic subunit, ilvB/ilvI-like; ~2-bp gap to PP_4679). The transcription order on the minus strand is therefore AHAS-large → AHAS-small → ilvC, a canonical ilvB(I)-ilvN(H)-ilvC cluster. This physically co-locates KARI with the AHAS enzyme that produces its substrates. KEGG annotations assign KO K00053 (KARI, EC 1.1.1.86); modules M00019 (Val/Ile biosynthesis) and M00570 (Ile biosynthesis from threonine); and pathways ppu00290 (Val/Leu/Ile biosynthesis), ppu00770 (pantothenate & CoA biosynthesis), ppu01210 (2-oxocarboxylic acid metabolism), and ppu01230 (amino-acid biosynthesis). The pantothenate/CoA link reflects that the KARI-DHAD product 2-ketoisovalerate is a branch-point precursor for pantothenate.

F007 — Sequence analysis predicts NADPH specificity and the two-Mg²⁺ acidic ligand cluster

Direct bioinformatic analysis of the Q88DZ0 sequence corroborates the annotation. (1) A canonical Rossmann dinucleotide-binding fingerprint GXGXXG is present at residues 23–28 (G23-Y-G25-S-Q-G28) in the N-domain. (2) The cofactor-specificity region immediately downstream (β2αB loop) contains basic residues Arg47 and Lys48 (…GLRKGSAT…, residues 45–52) plus Ser50/Thr52; positively charged residues at these positions are the structural hallmark that coordinates the 2′-phosphate of NADPH, indicating NADPH (not NADH) preference — consistent with the enzyme's formal name "Ketol-acid reductoisomerase (NADP(+))." (3) The C-terminal domain carries a conserved cluster of acidic residues — Asp190, Glu194, Glu226, Glu230 (all UniProt-annotated binding sites) — forming two Asp/Glu pairs that coordinate the two catalytic Mg²⁺ ions. (4) An annotated active-site residue His107 lies at the N/C-domain interface. This is grounded in the finding that specificity-loop residues determine cofactor preference in class I KARIs: "insertions in the specificity loops that confounded previous attempts to classify them according to loop length" (PMID: 25849365).

Mechanistic Model / Interpretation

Position in the branched-chain amino acid pathway

KARI (ilvC) is the second of four common enzymatic steps that build all three branched-chain amino acids from pyruvate (and, for isoleucine, 2-oxobutanoate). The pathway architecture in P. putida KT2440 is:

       pyruvate + pyruvate  (Val/Leu branch)
       pyruvate + 2-oxobutanoate  (Ile branch)
                 |
                 |  [ Step 1 ]  AHAS  (ilvBN / PP_4680-PP_4679)
                 v
(S)-2-acetolactate        OR   (S)-2-aceto-2-hydroxybutyrate
                 |
                 |  [ Step 2 ]  KARI  (ilvC / PP_4678)  <-- THIS GENE
                 |     Mg2+-dependent alkyl migration
                 |     + NADPH-dependent reduction
                 v
   (2R)-2,3-dihydroxy-isovalerate  OR  (2R,3R)-2,3-dihydroxy-3-methylvalerate
                 |
                 |  [ Step 3 ]  DHAD  (ilvD)  -- dehydration
                 v
2-ketoisovalerate       OR       2-keto-3-methylvalerate
     |                              |
     |  [ Step 4 ] transaminase     | [ Step 4 ] transaminase
     v                              v
  L-VALINE                     L-ISOLEUCINE
     |
     +--> (via ilvD product 2-ketoisovalerate)
          --> LEUCINE biosynthesis (leuABCD)
          --> PANTOTHENATE / CoA biosynthesis (panBCD)

KARI performs the pivotal chemistry that converts the acetohydroxy-acid intermediates into dihydroxy-acids. Its bifunctionality is remarkable: a single active site accommodates two mechanistically unrelated reactions. The isomerization step is an alkyl migration (methyl for the valine branch, ethyl for the isoleucine branch), positioning the substrate for the second reaction; this step requires the two active-site Mg²⁺ ions coordinated by the Asp190/Glu194 and Glu226/Glu230 pairs. The reduction step then uses hydride from NADPH bound in the N-terminal Rossmann domain.

Structure–function logic

Feature	Structural element (Q88DZ0)	Functional consequence
NAD(P)H binding	N-terminal Rossmann domain; GXGXXG at res 23–28	Binds nicotinamide cofactor for the reduction step
NADPH vs NADH selection	β2αB loop basic residues Arg47/Lys48	Coordinate 2′-phosphate of NADPH → NADPH preference
Two Mg²⁺ ions	Acidic cluster Asp190/Glu194/Glu226/Glu230 (C-domain)	Catalyze the Mg²⁺-dependent alkyl migration; position substrate
Active site	His107 at N/C-domain interface	Catalytic residue at the inter-domain cleft
Class I identity	338 aa, single subunit, KARI_prok signature	Compact bacterial enzyme (no C-domain duplication)
Induced fit	N/C-domain interface	Closes on Mg²⁺ + NADPH binding to assemble competent site

Localization and pathway integration

KARI is a soluble cytoplasmic enzyme — the entire BCAA biosynthetic pathway operates in the cytosol, with no signal peptide or membrane-targeting features. Its genomic clustering with AHAS (ilvBN) in KT2440 supports co-regulation and metabolic channeling of substrates directly from AHAS to KARI. Downstream, the shared intermediate 2-ketoisovalerate is a branch point not only for valine/leucine but also for pantothenate and coenzyme A biosynthesis, explaining KARI's assignment to pathway ppu00770 in addition to the BCAA pathways.

Note on catabolism versus biosynthesis

Much of the P. putida-specific historical literature (PMIDs 5030618, 4150713, 4150714, 4352175, 10217783, 19910413) concerns branched-chain amino acid catabolism (the bkd operon, branched-chain keto acid dehydrogenase, transaminases). These pathways degrade BCAAs and are distinct from the biosynthetic role of ilvC. KARI is unambiguously a biosynthetic enzyme; the catabolic literature is contextually relevant to P. putida BCAA metabolism but does not describe ilvC function directly.

Evidence Base

PMID	Title (abbrev.)	How it supports the findings
19362563	Conformational changes in a plant KARI upon Mg²⁺ and NADPH binding	Defines exact substrates/products and the two-reaction (alkyl migration + reduction) chemistry requiring Mg²⁺ and NADPH (F001)
23036858	Bacterial and plant KARIs have different mechanisms of induced fit	Establishes KARI as the second enzyme of BCAA biosynthesis; two-domain architecture with interface active site; taxonomic distribution absent in animals (F001, F002, F004)
25849365	Cofactor specificity motifs and induced fit in class I KARIs	Defines class I vs class II; induced-fit active-site closure on NAD(P)H binding; specificity-loop residues determine cofactor preference (F002, F003, F007)
21515217	Engineered KARI and ADH enable anaerobic isobutanol at theoretical yield	Shows NADPH specificity as wild-type default and KARI as engineerable biofuel node (F004)
36354071	M. tuberculosis KARI down-regulation affects persistence	Genetic evidence that KARI is important for bacterial fitness/persistence in vivo (F004)
24493249	FgIlv5 required for BCAA biosynthesis and virulence in F. graminearum	Deletion abolishes growth without Ile+Val; confirms biosynthetic role and phenotype of KARI loss (F004)
25081555	2-ketoisovalerate pathway enzymes in R. eutropha	Confirms the AHAS(ilvBH)→AHAIR(ilvC)→DHAD(ilvD) enzyme order and feedback regulation; contextual for F006
27600064	Poly(2-hydroxyisovalerate-co-lactate) biosynthesis in E. coli	Uses ilvCD alongside ilvBN for 2-hydroxyisovalerate production; contextual application of KARI

Supporting P. putida BCAA-catabolism literature (contextual, not describing ilvC directly): 5030618, 4150713, 4150714, 4352175, 10217783, 19910413.

Convergence of evidence

The functional assignment rests on four independent, converging evidence lines:
1. Enzymology/structure — primary literature defines KARI's reaction, cofactors, and mechanism.
2. Curated annotation — UniProt/HAMAP-Rule MF_00435 assigns EC 1.1.1.86, class I identity, sequence length, and catalytic residues specifically to Q88DZ0.
3. Genomic context — PP_4678 sits in a canonical ilvBN-ilvC cluster in KT2440.
4. Sequence analysis — the Rossmann fingerprint, NADPH-specificity loop residues, and two-Mg²⁺ acidic ligand cluster are all identifiable in the Q88DZ0 primary sequence.

Limitations and Knowledge Gaps

No direct experimental characterization of the P. putida KT2440 enzyme. All mechanistic and structural detail is inferred from orthologues (plant, E. coli, M. tuberculosis, R. eutropha, F. graminearum) and from bioinformatic/curated annotation. There is no published crystal structure, kinetic study, or knockout phenotype specifically for Q88DZ0/PP_4678.
Cofactor specificity is predicted, not measured. NADPH preference for Q88DZ0 is inferred from the β2αB loop basic residues (Arg47/Lys48) and the enzyme name. Direct kinetic determination of NADPH vs NADH preference for this specific enzyme has not been done.
Substrate-branch kinetics unknown. The relative activity toward the valine-branch (2-acetolactate) versus isoleucine-branch (2-aceto-2-hydroxybutyrate) substrate has not been measured for this enzyme; branch partitioning is assumed to follow the general KARI paradigm.
Residue numbering caveats. Active-site (His107) and metal-ligand residue positions are drawn from UniProt annotation and sequence analysis; they have not been validated by mutagenesis in this organism.
Regulation not experimentally defined. The genomic clustering with ilvBN implies co-regulation, but promoter mapping, transcriptional regulation, and feedback inhibition of KARI in KT2440 specifically remain uncharacterized (though feedback inhibition of KARI/AHAIR by BCAAs is documented in related bacteria such as R. eutropha and C. glutamicum).

Proposed Follow-up Experiments / Actions

Recombinant expression and steady-state kinetics. Express Q88DZ0 (His-tagged) in E. coli, purify, and determine k_cat/K_M for both branch substrates with NADPH and NADH; confirm the predicted strong NADPH preference and quantify the NADH/NADPH selectivity ratio.
Metal and cofactor dependence. Measure Mg²⁺ (and Mn²⁺ substitution) dependence and stoichiometry by ITC; verify the two-metal requirement and the roles of Asp190/Glu194/Glu226/Glu230 via alanine mutagenesis.
Structural determination. Solve the crystal structure (or generate an AlphaFold model followed by validation) of Q88DZ0 with bound Mg²⁺/NADPH to confirm the class I two-domain fold, the interface active site, and induced-fit closure.
Genetic knockout in KT2440. Delete PP_4678 and test for BCAA auxotrophy (growth rescue by valine + isoleucine), confirming the essential biosynthetic role in situ.
Operon/transcription mapping. Define the PP_4680–PP_4679–PP_4678 transcript(s) by RT-PCR/RNA-seq and identify the promoter and any BCAA-responsive regulation, testing co-regulation with AHAS.
Metabolic-engineering assessment. Evaluate Q88DZ0 (wild-type and NADH-swapped variants) as a KARI module for isobutanol / 2-ketoisovalerate-derived product biosynthesis in P. putida, exploiting the organism's robust solvent tolerance.

Conclusion

ilvC (Q88DZ0, PP_4678) of Pseudomonas putida KT2440 encodes ketol-acid reductoisomerase (KARI/AHAIR, EC 1.1.1.86), a soluble cytoplasmic, 338-residue class I enzyme that catalyzes the second step of branched-chain amino acid biosynthesis. Using two Mg²⁺ ions and NADPH in a single active site, it performs an alkyl-migration isomerization followed by reduction, converting (S)-2-acetolactate to (2R)-2,3-dihydroxyisovalerate (valine/leucine branch) and (S)-2-aceto-2-hydroxybutyrate to (2R,3R)-2,3-dihydroxy-3-methylvalerate (isoleucine branch). It acts downstream of acetohydroxyacid synthase (ilvBN, encoded immediately upstream) and upstream of dihydroxyacid dehydratase (ilvD), also feeding pantothenate/CoA biosynthesis. The assignment is supported by convergent enzymological, curated-annotation, genomic-context, and sequence-analysis evidence, though no experiment has yet been performed on the KT2440 enzyme itself.

Artifacts

Citations

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📄 View Raw YAML

id: Q88DZ0
gene_symbol: ilvC
product_type: PROTEIN
status: DRAFT
taxon:
  id: NCBITaxon:160488
  label: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950
    / KT2440)
description: ilvC encodes NADP-dependent ketol-acid reductoisomerase, a Mg-dependent
  enzyme in branched-chain amino acid biosynthesis.
existing_annotations:
- term:
    id: GO:0000287
    label: magnesium ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000104
  review:
    summary: Magnesium binding is a required cofactor feature but not the specific
      gene function.
    action: KEEP_AS_NON_CORE
    reason: UniProt indicates two Mg(2+) ions per subunit for IlvC; this supports
      retention as non-core cofactor information.
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
      supporting_text: Binds 2 magnesium ions per subunit
    - reference_id: file:PSEPK/ilvC/ilvC-goa.tsv
      supporting_text: "GO:0000287\tmagnesium ion binding"
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
      supporting_text: IlvC requires **NADPH** for the reduction half-reaction and
        a **divalent metal ion (most commonly Mg²⁺)** to support the alkyl-migration/isomerization
        chemistry, which is mechanistically integrated in a single catalytic cycle
        for most KARIs.
  qualifier: enables
- term:
    id: GO:0004455
    label: ketol-acid reductoisomerase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: Ketol-acid reductoisomerase activity is the specific catalytic function
      of IlvC.
    action: ACCEPT
    reason: The reviewed UniProt entry assigns EC 1.1.1.86 and describes the alkyl-migration/reduction
      reaction in BCAA biosynthesis.
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
      supporting_text: Catalyzes an alkyl-migration followed by a ketol-acid reduction
    - reference_id: file:PSEPK/ilvC/ilvC-goa.tsv
      supporting_text: "GO:0004455\tketol-acid reductoisomerase activity"
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
      supporting_text: Ketol-acid reductoisomerase (KARI; IlvC/AHAIR)** is a conserved
        bacterial enzyme family within the 6-phosphogluconate dehydrogenase (6PGDH)-type
        superfamily that performs a chemically coupled **alkyl-migration (isomerization)**
        and **NADP(H)-dependent reduction** step in BCAA biosynthesis.
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-openscientist.md
      supporting_text: >-
        The gene **_ilvC_** (ordered locus **PP_4678**; UniProt **Q88DZ0**) of
        *Pseudomonas putida* strain KT2440 (ATCC 47054 / DSM 6125 / NCIMB 11950)
        encodes **ketol-acid reductoisomerase (KARI)**
  qualifier: enables
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IEA
  original_reference_id: GO_REF:0000118
  review:
    summary: Cytosol is plausible cellular context but not the defining function.
    action: KEEP_AS_NON_CORE
    reason: The TreeGrafter cytosol annotation is consistent with a soluble bacterial
      biosynthetic enzyme but should remain non-core. Falcon deep research supports
      cytosolic localization by homology, noting no KT2440-specific localization experiment
      was retrieved.
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-goa.tsv
      supporting_text: "GO:0005829\tcytosol"
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
      supporting_text: a Gram-negative bacterial IlvC homolog was reported as a **soluble
        protein** that could be purified without detergents, consistent with a **cytosolic
        enzyme** (as expected for a central-metabolism biosynthetic enzyme operating
        on cytosolic intermediates).
  qualifier: located_in
- term:
    id: GO:0009082
    label: branched-chain amino acid biosynthetic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: Branched-chain amino acid biosynthetic process is true but less specific
      than the valine/isoleucine branch terms.
    action: MARK_AS_OVER_ANNOTATED
    reason: IlvC acts at a shared BCAA pathway step; the specific L-valine annotation
      and missing isoleucine annotation are more informative.
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
      supporting_text: Involved in the biosynthesis of branched-chain amino acids
    - reference_id: file:PSEPK/ilvC/ilvC-goa.tsv
      supporting_text: "GO:0009082\tbranched-chain amino acid biosynthetic process"
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
      supporting_text: IlvC is the second step in the pyruvate-to-2-ketoisovalerate
        segment of **branched-chain amino-acid biosynthesis**. It supports synthesis
        of valine and isoleucine directly and leucine indirectly via 2-oxoisovalerate-derived
        metabolism.
  qualifier: involved_in
- term:
    id: GO:0009099
    label: L-valine biosynthetic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: IlvC participates directly in L-valine biosynthesis.
    action: ACCEPT
    reason: UniProt maps IlvC to the L-valine biosynthesis route from pyruvate, step
      2 of 4. Falcon deep research provides KT2440-specific genetic evidence (conditional
      essentiality on minimal medium and BCAA auxotrophy of ilvC mutants).
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
      supporting_text: 'L-valine from pyruvate: step 2/4'
    - reference_id: file:PSEPK/ilvC/ilvC-goa.tsv
      supporting_text: "GO:0009099\tL-valine biosynthetic process"
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
      supporting_text: A genome-wide knockout screen on glucose minimal medium identified
        **ilvC (PP4678)** among genes whose disruption prevents growth on M9 minimal
        medium, i.e., conditionally essential in that environment.
  qualifier: involved_in
- term:
    id: GO:0016491
    label: oxidoreductase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: Oxidoreductase activity is a broad parent of the specific ketol-acid
      reductoisomerase function.
    action: MARK_AS_OVER_ANNOTATED
    reason: The specific EC-linked ketol-acid reductoisomerase annotation should be
      preferred over the broad oxidoreductase parent.
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
      supporting_text: EC=1.1.1.86
    - reference_id: file:PSEPK/ilvC/ilvC-goa.tsv
      supporting_text: "GO:0016491\toxidoreductase activity"
  qualifier: enables
- term:
    id: GO:0050661
    label: NADP binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: NADP binding is a cofactor feature of IlvC.
    action: KEEP_AS_NON_CORE
    reason: The reaction uses NADP/NADPH, but binding alone is less informative than
      the enzyme activity.
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
      supporting_text: NADP(+)
    - reference_id: file:PSEPK/ilvC/ilvC-goa.tsv
      supporting_text: "GO:0050661\tNADP binding"
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
      supporting_text: IlvC requires **NADPH** for the reduction half-reaction and
        a **divalent metal ion (most commonly Mg²⁺)** to support the alkyl-migration/isomerization
        chemistry, which is mechanistically integrated in a single catalytic cycle
        for most KARIs.
  qualifier: enables
- term:
    id: GO:1901705
    label: L-isoleucine biosynthetic process
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: IlvC also participates directly in isoleucine biosynthesis.
    action: NEW
    reason: The reviewed UniProt entry has an L-isoleucine biosynthesis pathway statement,
      but the fetched GOA table lacks this specific process annotation.
      Falcon deep research independently reports isoleucine auxotrophy of KT2440 ilvC
      mutants, supporting a direct role in isoleucine biosynthesis.
    supported_by:
    - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
      supporting_text: 'L-isoleucine from 2-oxobutanoate: step 2/4'
    - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
      supporting_text: '*ilvC* mutants showed branched-chain amino-acid auxotrophy,
        with reported requirements involving isoleucine and valine/leucine supplementation
        depending on the assay context.'
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO
    terms
  findings: []
- 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: file:PSEPK/ilvC/ilvC-uniprot.txt
  title: UniProtKB reviewed entry for ilvC
  findings:
  - statement: UniProt provides the reviewed functional description used for the ilvC
      review.
- id: file:PSEPK/ilvC/ilvC-goa.tsv
  title: QuickGO GOA annotations for ilvC
  findings:
  - statement: The fetched GOA table contains the annotations reviewed for ilvC.
- id: file:interpro/panther/PTHR21371/PTHR21371-metadata.yaml
  title: PANTHER family metadata for ilvC
  findings:
  - statement: PANTHER places IlvC in the ketol-acid reductoisomerase family.
- id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
  title: Falcon (Edison Scientific) deep research report for P. putida KT2440 ilvC
    (Q88DZ0 / PP_4678)
  findings:
  - supporting_text: Ketol-acid reductoisomerase (KARI; IlvC/AHAIR)** is a conserved
      bacterial enzyme family within the 6-phosphogluconate dehydrogenase (6PGDH)-type
      superfamily that performs a chemically coupled **alkyl-migration (isomerization)**
      and **NADP(H)-dependent reduction** step in BCAA biosynthesis.
  - supporting_text: IlvC/AHAIR is the **second** enzyme in the pyruvate-to-2-ketoisovalerate
      segment (AHAS → AHAIR/IlvC → DHAD/IlvD).
  - supporting_text: IlvC requires **NADPH** for the reduction half-reaction and a
      **divalent metal ion (most commonly Mg²⁺)** to support the alkyl-migration/isomerization
      chemistry, which is mechanistically integrated in a single catalytic cycle for
      most KARIs.
  - supporting_text: a Gram-negative bacterial IlvC homolog was reported as a **soluble
      protein** that could be purified without detergents, consistent with a **cytosolic
      enzyme** (as expected for a central-metabolism biosynthetic enzyme operating
      on cytosolic intermediates).
  - supporting_text: A genome-wide knockout screen on glucose minimal medium identified
      **ilvC (PP4678)** among genes whose disruption prevents growth on M9 minimal
      medium, i.e., conditionally essential in that environment.
  - supporting_text: '*ilvC* mutants showed branched-chain amino-acid auxotrophy,
      with reported requirements involving isoleucine and valine/leucine supplementation
      depending on the assay context.'
- id: file:PSEPK/ilvC/ilvC-deep-research-openscientist.md
  title: OpenScientist literature and database synthesis for PSEPK IlvC
  findings:
  - statement: >-
      Confirms Q88DZ0 as the ketol-acid reductoisomerase used at the shared second
      step of branched-chain amino-acid biosynthesis.
    supporting_text: >-
      The gene **_ilvC_** (ordered locus **PP_4678**; UniProt **Q88DZ0**) of
      *Pseudomonas putida* strain KT2440 (ATCC 47054 / DSM 6125 / NCIMB 11950)
      encodes **ketol-acid reductoisomerase (KARI)**
  - statement: >-
      No direct Q88DZ0 experiment was found; target-specific kinetics, structure,
      localization, and regulation therefore remain unverified.
    supporting_text: >-
      No direct experimental characterization of the *P. putida* KT2440 enzyme.
core_functions:
- description: Mg-dependent NADP ketol-acid reductoisomerase catalyzing a shared valine/isoleucine
    biosynthetic step.
  molecular_function:
    id: GO:0004455
    label: ketol-acid reductoisomerase activity
  directly_involved_in:
  - id: GO:0009099
    label: L-valine biosynthetic process
  - id: GO:1901705
    label: L-isoleucine biosynthetic process
  supported_by:
  - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
    supporting_text: Catalyzes an alkyl-migration followed by a ketol-acid reduction
  - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
    supporting_text: 'L-valine from pyruvate: step 2/4'
  - reference_id: file:PSEPK/ilvC/ilvC-uniprot.txt
    supporting_text: 'L-isoleucine from 2-oxobutanoate: step 2/4'
  - reference_id: file:PSEPK/ilvC/ilvC-deep-research-falcon.md
    supporting_text: IlvC/AHAIR is the **second** enzyme in the pyruvate-to-2-ketoisovalerate
      segment (AHAS → AHAIR/IlvC → DHAD/IlvD).
  - reference_id: file:PSEPK/ilvC/ilvC-deep-research-openscientist.md
    supporting_text: >-
      The gene **_ilvC_** (ordered locus **PP_4678**; UniProt **Q88DZ0**) of
      *Pseudomonas putida* strain KT2440 (ATCC 47054 / DSM 6125 / NCIMB 11950)
      encodes **ketol-acid reductoisomerase (KARI)**
proposed_new_terms: []
suggested_questions:
- question: Does KT2440 IlvC show physiologically meaningful differences in activity
    toward acetolactate versus acetohydroxybutyrate substrates?
suggested_experiments:
- description: Measure purified IlvC kinetics with valine- and isoleucine-branch ketol-acid
    substrates and test ilvC knockout rescue by branched-chain amino acid supplementation.
  experiment_type: enzyme kinetics and genetic complementation

ilvC

Gene Description

Existing Annotations Review

Core Functions

Mg-dependent NADP ketol-acid reductoisomerase catalyzing a shared valine/isoleucine biosynthetic step.

References

Suggested Questions for Experts

Suggested Experiments

Deep Research

Falcon

Research report: Functional annotation of Pseudomonas putida KT2440 ilvC (UniProt Q88DZ0; locus PP_4678)

0) Target verification (critical disambiguation)

1) Key concepts and current understanding

1.1 Definitions and enzyme class

1.2 Reaction and pathway position

1.3 Substrate specificity and promiscuity

1.4 Cofactor dependence (NADPH) and metal requirement

1.5 Cellular localization

2) Pseudomonas putida KT2440-specific functional evidence

2.1 Conditional essentiality and auxotrophy phenotypes

2.2 Integration in KT2440 redox and carbon-flux engineering

3) Recent developments (prioritizing 2023–2024)

3.1 2023: Tunable growth/product “metabolic valve” in P. putida KT2440

3.2 2024: Authoritative review synthesis for isobutanol pathways

3.3 2024: Cofactor rewiring of IlvC to shift NADPH → NADH usage

4) Current applications and real-world implementations

4.1 Industrial biotechnology: BCAA-pathway-derived chemicals in P. putida

4.2 Systems metabolic engineering: growth-coupled production control (2023)

5) Expert opinions / analysis (authoritative synthesis)

5.1 IlvC as a predictable redox bottleneck

5.2 Practical interpretation for KT2440 functional annotation

6) Key quantitative statistics and data points

7) Consolidated functional annotation (recommended)

8) Limitations of this evidence set

Artifacts

Citations

OpenScientist

Functional Annotation Report: ilvC / Ketol-Acid Reductoisomerase (Q88DZ0) in Pseudomonas putida KT2440

Summary

Key Findings

F001 — ilvC encodes ketol-acid reductoisomerase, the second step of BCAA biosynthesis

F002 — KARI is an Mg²⁺/NADPH-dependent, two-domain enzyme using an induced-fit mechanism

F003 — Q88DZ0 is a class I / "type 1" prokaryotic KARI

F004 — KARI operates in the cytoplasm; it is a validated antimicrobial target absent from animals and a key biofuel-engineering node

F005 — Protein-specific annotation: 338-residue two-domain enzyme, 2 Mg²⁺/subunit, step 2/4 of both Val and Ile routes

F006 — PP_4678 is genomically clustered with acetohydroxyacid synthase genes and feeds Val/Ile and pantothenate/CoA pathways

F007 — Sequence analysis predicts NADPH specificity and the two-Mg²⁺ acidic ligand cluster

Mechanistic Model / Interpretation

Position in the branched-chain amino acid pathway

Structure–function logic

Localization and pathway integration

Note on catabolism versus biosynthesis

Evidence Base

Convergence of evidence

Limitations and Knowledge Gaps

Proposed Follow-up Experiments / Actions

Conclusion

Artifacts

Citations

📄 View Raw YAML