| Feature | Summary for *Pseudomonas putida* KT2440 VanA (UniProt Q88GI6; gene **vanA**; locus **PP_3736**) | Supporting citation(s) |
|---|---|---|
| Identity verification | The target is **VanA/PP_3736** from *P. putida* KT2440, annotated as the oxygenase component of **vanillate O-demethylase**; the partner gene is **vanB/PP_3737**. A pathway figure for KT2440 explicitly places **vanA/vanB** at the **vanillate (vanillic acid) → protocatechuate (protocatechuic acid)** step. | (pqac-00000005, pqac-00000010, pqac-00000013) |
| Primary biochemical function | VanA is the **terminal oxygenase** of the two-component **VanAB** vanillate O-demethylase system that catalyzes oxidative demethylation of vanillate to protocatechuate, releasing **formaldehyde** as a coproduct. | (pqac-00000003, pqac-00000000) |
| Enzyme class / mechanism | VanAB is described as a **Rieske non-heme iron monooxygenase** / Rieske-type aromatic O-demethylase. The chemistry is an **oxidative O-demethylation** rather than a THF-dependent methyl-transfer route. | (pqac-00000000, pqac-00000004, pqac-00000006) |
| Reaction | Canonical reaction in KT2440: **vanillate + reducing equivalents + O2 → protocatechuate + formaldehyde** (exact stoichiometric balancing varies by assay framing, but all cited sources agree on vanillate-to-protocatechuate conversion with formaldehyde release). | (pqac-00000000, pqac-00000001, pqac-00000003) |
| Cofactors / metal centers | VanA-family oxygenases contain a **Rieske [2Fe-2S] cluster** and a **non-heme iron** catalytic center; electron transfer is supplied through the reductase partner and can draw on **NADH and/or NADPH** in assays. | (pqac-00000002, pqac-00000004, pqac-00000001) |
| Partner subunit VanB | **VanB** is the reductase component of the two-component system. General VanAB descriptions assign VanB **FMN-, NADPH-, and [2Fe-2S]-binding features** and the role of delivering electrons to VanA for catalysis. In KT2440 literature, VanB is explicitly the reductase for vanillate O-demethylase. | (pqac-00000004, pqac-00000006) |
| Subunit architecture | VanAB is a **two-component** system, commonly described as **VanA (oxygenase) + VanB (reductase)**; one study further refers to it as a **heterodimeric** system in the context of *Pseudomonas* vanillate O-demethylase. | (pqac-00000003, pqac-00000002) |
| Substrate specificity: confirmed core substrate | The best-supported physiological substrate in KT2440 is **vanillate**. VanAB enables growth on vanillate as a sole carbon/energy source by converting it to protocatechuate, which then enters central aromatic catabolism. | (pqac-00000000, pqac-00000006, pqac-00000013) |
| Substrate specificity: broader/promiscuous activity | Across homologous VanAB systems, activity extends to **meta-methoxylated aromatic acids** and sometimes compounds such as **veratrate** and **syringate/3MGA**, but the evidence indicates **clear preference for vanillate** (and in some systems syringate) over **3MGA**. For KT2440 specifically, recent comparative work notes slower syringate conversion and no in vivo 3MGA O-demethylation despite in vitro activity toward 3MGA. | (pqac-00000002, pqac-00000004, pqac-00000007, pqac-00000012) |
| Product and byproduct | Main aromatic product is **protocatechuate**; one-carbon byproduct is **formaldehyde**, creating a need for detoxification or assimilation capacity during growth/engineering. | (pqac-00000000, pqac-00000003, pqac-00000009) |
| Pathway role | VanA operates in the **upper funneling pathway for lignin-derived guaiacyl aromatics**, especially vanillate produced from compounds such as ferulate/vanillin. The product **protocatechuate** feeds into the **β-ketoadipate/pca pathway**. | (pqac-00000005, pqac-00000013, pqac-00000000) |
| Physiological importance in KT2440 | Native VanAB supports **growth on vanillate** and is central to lignin-aromatic funneling. In adaptive laboratory evolution and pathway-engineering experiments, boosting VanAB function improved vanillate utilization and aromatic catabolism. | (pqac-00000006, pqac-00000000) |
| Genetic context / operon | In KT2440, **vanA and vanB are organized as a vanAB operon**. Multiple studies discuss deletion or constitutive re-expression of this operon in engineering backgrounds. | (pqac-00000000, pqac-00000003, pqac-00000006) |
| Regulation | Evidence summarized from recent comparative work indicates regulation by **VanR**, a **negative transcriptional regulator** relieved by **vanillate** as inducer/effector. ALE studies in KT2440 also identified beneficial mutations in regulators and formaldehyde-detox genes linked to improved VanAB-dependent growth. | (pqac-00000002, pqac-00000006, pqac-00000008) |
| Cellular localization | No direct experimental localization for KT2440 VanA was recovered in the gathered sources. Given its classification as a bacterial **Rieske non-heme iron oxygenase** with no evidence here for secretion or membrane anchoring, the most defensible annotation from current evidence is **intracellular/cytosolic aromatic catabolism** rather than extracellular function. | (pqac-00000000, pqac-00000004) |
| Formaldehyde coupling / detoxification | VanAB function is tightly coupled to **formaldehyde detoxification**. In heterologous expression experiments, formate accumulation began as protocatechuate formed, and loss of **frmA** sharply reduced formate production and impaired conversion, showing the burden imposed by VanAB-derived formaldehyde. In KT2440 ALE, mutations in **fghA** and related loci were selected in VanAB backgrounds. | (pqac-00000009, pqac-00000006, pqac-00000008) |
| Quantitative assay data | In *E. coli* expressing *P. putida* **vanAB**, lysate assays produced **15.5 ± 2.2 mM protocatechuate after 3 h** with reported specific activity **0.88 μmol protocatechuate min−1 g−1 whole-cell extract**; **5 mM vanillate** was used in whole-cell assays, and both **NADH/NADPH** supported activity. | (pqac-00000001) |
| Quantitative physiology / engineering data in KT2440 | In a 2024 KT2440 study, evolved strains relying on native **VanAB** showed **~1.8-fold faster growth** than THF-dependent demethylase strains, and combining top mutations yielded **~5-fold faster vanillate consumption during the first 8 h** versus wild type. | (pqac-00000006) |
| Quantitative relevance to production strain design | Deleting **vanAB** is a standard strategy when the goal is to **accumulate vanillin/vanillate-derived products** rather than consume them. Example: a 2025 KT2440-derived vanillin process explicitly included **vanAB deletion**; with in situ product recovery, total vanillin recovery reached **3.35 g/L** from ferulic acid. | (pqac-00000000, pqac-00000006) |
| Real-world / biotechnological applications | VanA is important in **lignin valorization**, where microbial strains are engineered either to **enhance O-demethylation/funneling** (improving assimilation of methoxylated aromatics) or to **disable vanAB** to accumulate upstream products such as vanillin or route flux to polymer precursors such as β-ketoadipate/muconate. | (pqac-00000000, pqac-00000006, pqac-00000007) |
| Key references (year / DOI / URL) | **Bleem et al., 2024**, *Metabolic Engineering*, doi: **10.1016/j.ymben.2024.06.009**, https://doi.org/10.1016/j.ymben.2024.06.009; **Hibi et al., 2005**, *FEMS Microbiol Lett.*, doi: **10.1016/j.femsle.2005.09.036**, https://doi.org/10.1016/j.femsle.2005.09.036; **García-Hidalgo et al., 2020**, *Appl Environ Microbiol*, doi: **10.1128/AEM.02442-19**, https://doi.org/10.1128/AEM.02442-19; broader VanAB context: **Donoso et al., 2022**, doi: **10.3390/microorganisms11010078**, https://doi.org/10.3390/microorganisms11010078. | (pqac-00000006, pqac-00000001, pqac-00000005, pqac-00000004) |


*Table: This table summarizes the functional annotation of VanA (Q88GI6/PP_3736) in Pseudomonas putida KT2440, covering identity, reaction, cofactors, pathway context, regulation, quantitative data, and engineering relevance. Each row is explicitly tied to the gathered evidence contexts for direct traceability.*