| Target | Enzyme name / function | EC number | Reaction step in tryptophan biosynthesis | Gene organization / genomic context | Key experimental evidence in *P. putida* KT2440 | Growth complementation / quantitative conditions | Key citation(s) |
|---|---|---|---|---|---|---|---|
| **trpF** (UniProt **Q88LE0**; locus **PP_1995**) | **Phosphoribosyl anthranilate isomerase** / **N-(5'-phosphoribosyl)anthranilate isomerase**; enzyme assigned to the tryptophan branch from anthranilate toward indole-3-glycerol phosphate (pqac-00000002, pqac-00000003) | **EC 5.3.1.24** reported for TrpF/PRAI in general pathway annotations and genome annotations cited in retrieved literature; KT2440 paper functionally assigns PP_1995 as phosphoribosyl anthranilate isomerase (pqac-00000002) | Catalyzes the **PRAI step** after anthranilate phosphoribosyltransferase (**TrpD**) and before indole-3-glycerol phosphate synthase (**TrpC**); thus it converts the phosphoribosyl-anthranilate intermediate within the anthranilate → indole-3-glycerol phosphate segment of the pathway (pqac-00000002, pqac-00000000) | **Monocistronic**, **unlinked** to the other main trp clusters; trp genes in KT2440 are distributed in separate regions, with **trpBA** and **trpGDC** in operons while **trpF** is a **single transcriptional unit**. In sequenced *Pseudomonas* spp., trpF is consistently flanked by **truA** and **accD**. In KT2440, the upstream ORF (**PP1994**) is **61 nt** away and the downstream ORF (**PP1996**) starts **222 nt** after the PP_1995 stop codon; RT-PCR for cotranscription was negative, supporting that **trpF is most probably a single cistron** (pqac-00000001, pqac-00000002, pqac-00000003) | Because spontaneous mutants were not recovered, the authors made a **targeted chromosomal knockout** using a **pCHESI/pCHESIWKm site-specific homologous inactivation strategy**. An internal fragment of about **500 bp** was amplified with primers **TrpF-XbaI** and **TrpF-2**, cloned, introduced by **electroporation**, and confirmed by **colony PCR** and **Southern blotting**. The resulting trpF-deficient clones were **tryptophan auxotrophs**, with only **very small colonies** appearing without tryptophan supplementation (pqac-00000001, pqac-00000004) | In feeding assays on **M9 minimal medium with 16 mM citrate** as carbon source, supplements were added to **0.2 mM final concentration** (except tryptophan at **0.6 mM** for the trpA assay comparison). The **TrpF mutant** showed growth pattern: **M9 +/-**, **+ tryptophan: +**, **+ chorismate: +/-**, **+ anthranilate: +/-**, **+ indole: +**. This indicates rescue by **tryptophan** or **indole**, but not effective rescue by **anthranilate**, consistent with TrpF acting downstream of anthranilate formation and upstream of indole production (pqac-00000001, pqac-00000005) | Molina-Henares et al., **2009-12**, *Microbial Biotechnology*, DOI: **10.1111/j.1751-7915.2008.00062.x**, URL: **https://doi.org/10.1111/j.1751-7915.2008.00062.x** (pqac-00000001, pqac-00000002, pqac-00000003, pqac-00000004, pqac-00000005); pathway/regulatory context from Matulis et al., **2022-04**, *Int. J. Mol. Sci.*, DOI: **10.3390/ijms23094649**, URL: **https://doi.org/10.3390/ijms23094649** (pqac-00000000) |


*Table: This table summarizes the validated functional annotation of Pseudomonas putida KT2440 trpF/PP_1995, including its enzymatic role, pathway position, genomic organization, and direct mutant evidence. It is useful as a concise evidence map linking the locus to tryptophan biosynthesis and experimentally observed auxotrophy/complementation.*