Organism: Pseudomonas putida KT2440 (strain ATCC 47054 / DSM 6125 / NCIMB 11950; taxid 160488)
Gene / locus: PP_3157 (OrderedLocusName; EMBL AAN68765.1)
UniProt: Q88I44 (Q88I44_PSEPK), 263 aa, monomer, protein existence level 3 (inferred from homology)
EC: 3.1.3.15 — histidinol-phosphatase
The requested target is PP_3157 / Q88I44 in Pseudomonas putida KT2440. All authoritative annotations are internally consistent and match the target:
ppu:PP_3157; STRING 160488.PP_3157; BioCyc PPUT160488:G1G01-3376-MONOMER).There is no gene-symbol ambiguity concern here: "PP_3157" is an unambiguous ordered-locus identifier unique to the P. putida KT2440 genome. The functional annotation below is supported by strong homology and by direct experimental/structural characterization of close orthologs; the specific PP_3157 protein has not itself been enzymatically assayed (annotation is inferred from homology, PE3).
PP_3157 encodes a histidinol-phosphate phosphatase (HolPase, EC 3.1.3.15) of the inositol-monophosphatase (IMPase)–like "HisN" family — the third, evolutionarily independent HolPase family (distinct from the HAD-type HisB of E. coli and the PHP-type of Bacillus). Acting in the cytoplasm as a soluble, Mg²⁺-dependent phosphomonoesterase, it catalyzes the penultimate step of L-histidine biosynthesis: hydrolysis of L-histidinol-phosphate to L-histidinol + inorganic phosphate. The product L-histidinol is then oxidized by histidinol dehydrogenase (HisD) to L-histidine.
Reaction (UniProt catalytic activity):
L-histidinol phosphate + H₂O → L-histidinol + phosphate (EC 3.1.3.15)
Pathway position (UniProt): Amino-acid biosynthesis; L-histidine biosynthesis; L-histidine from 5-phospho-α-D-ribose-1-diphosphate: step 8 of 9. This is the penultimate phosphomonoester-hydrolysis step; it removes the phosphate installed earlier (by the aminotransferase HisC on imidazole-acetol phosphate → histidinol-phosphate) to generate the free alcohol L-histidinol [PMID 26994138].
Family context — three unrelated HolPase families. The dephosphorylation of histidinol-phosphate is performed by three non-homologous enzyme families across life: HAD-type (Gammaproteobacteria such as E. coli/Salmonella, as the N-terminal domain of bifunctional HisB), PHP-type (yeast and Firmicutes such as Bacillus subtilis), and the IMPase-like "HisN" family present in Actinobacteria (e.g., Corynebacterium glutamicum HisN) and plants [PMID 28720084]. PP_3157's InterPro/Pfam/TIGRFAM signatures place it squarely in the IMPase-like family — notable because P. putida is a Gammaproteobacterium yet uses the IMPase-type rather than the enterobacterial HAD-type HolPase (the reason is explained by comparative genomics in §6b: Pseudomonas has a monofunctional IGP dehydratase and no HAD-type HolPase domain).
Although this family is structurally homologous to myo-inositol monophosphatases (IMPases), its members are specific for L-histidinol-phosphate and do not dephosphorylate D-myo-inositol-1-phosphate, the canonical IMPase substrate. This was shown biochemically for the orthologous plant enzyme MtHPP (Medicago truncatula):
"MtHPP was able to cleave inorganic phosphate from HOLP but not from d-myo-inositol-1-phosphate, the main substrate of IMPases." [PMID 26994138]
Structurally, specificity is conferred by the C-terminal domain (which shares little identity with true IMPases), while the catalytic machinery resides in the conserved N-terminal domain [PMID 26994138]. Importantly, several IMPase-superfamily paralogs that are sequence-similar to HisN — CysQ, ImpA (SuhB) — are not HolPases and do not participate in histidine biosynthesis [PMID 28720084]; correct annotation therefore depends on the specific HisN/TIGR02067 signature carried by PP_3157 rather than generic IMPase homology.
Cofactor: Mg²⁺ (UniProt COFACTOR: Mg(2+); ChEBI:18420). The reaction of IMPase-type HolPases "requires Mg(2+) cations, is catalyzed mainly by amino acid residues from the N-terminal domain" [PMID 26994138], consistent with the classic multi-Mg²⁺ IMPase phosphomonoesterase mechanism.
Metal-binding residues (UniProt features, PIRSR): five annotated Mg²⁺-coordinating positions — 74, 90, 92, 93, 214 (positions 74, 92, 214 flagged "catalytic"). Residues 90–95 form the diagnostic IMPase metal-binding motif "DPIDGT" found in the PP_3157 sequence (…VLDPIDGTRAF…), the hallmark of the inositol-monophosphatase fold.
Direct sequence evidence on PP_3157 itself. Independent motif analysis of the 263-aa Q88I44 sequence confirms an intact IMPase active site: the PROSITE inositol-monophosphatase signature-1 (PS00629) matches as WVLDPIDGTRAF at residues 87–98, and the UniProt-annotated metal ligands map to acidic residues Glu74, Asp90, Asp93, and Asp214. An aromatic-rich active-site segment (…MRYGGDCYAYCM…, res ~185–196) supplies the conserved aromatic residues that, together with a catalytic aspartate, are required for activity in this family. Mutational work on the C. glutamicum family identified a "highly conserved aspartate residue accompanied by several aromatic amino acid residues" as essential for activity [PMID 28720084], matching PP_3157's architecture. The protein has no signal peptide or transmembrane region, consistent with a soluble cytoplasmic enzyme.
Oligomeric state: IMPase-superfamily enzymes typically function as homodimers (the plant MtHPP is a dimer [PMID 26994138]); PP_3157 is annotated as a single 263-aa chain and is expected to assemble similarly.
The enzyme is a soluble cytoplasmic protein with no signal peptide or transmembrane segments; histidine biosynthesis occurs in the bacterial cytoplasm. The compartmentalization of this family tracks the site of His biosynthesis: in plants the orthologous IMPase-like HolPase is targeted to the plastid stroma, where the pathway operates:
"an IMPL2:green fluorescent protein fusion protein was targeted to the plastid, where His biosynthesis occurs in plants." [PMID 20023146]
For a bacterium such as P. putida, the corresponding compartment is the cytoplasm.
KEGG genome mapping of P. putida KT2440 confirms and sharpens the assignment:
PP_3157 supplies L-histidinol to the terminal oxidation steps of the his pathway. Its product is the substrate of histidinol dehydrogenase (HisD), which performs the two NAD⁺-dependent oxidations converting L-histidinol → L-histidinaldehyde → L-histidine. Thus PP_3157 sits between the transaminase step (HisC) and the final dehydrogenase step (HisD) in the linear, feedback-regulated histidine biosynthetic pathway (whose genes are dispersed across the P. putida genome rather than in a single operon; §6b). The pathway is metabolically expensive and tightly regulated; loss of the HolPase step blocks de novo histidine production.
Genetic validation of the family's role. The physiological (in vivo) HolPase identity of this IMPase-like family is established by cross-species complementation: an Arabidopsis IMPase-like protein rescued a bacterial hisN mutant —
"Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant." [PMID 20023146]
and deletion/knockout studies of hisN in Actinobacteria confirmed its requirement for histidine prototrophy, with the caveat that genome-encoded paralogs can provide partial redundancy [PMID 28720084, PMID 16901339].
Because PP_3157 has not itself been assayed, the reliability of the annotation depends on how closely it resembles enzymes that have been characterized. Global pairwise alignment (Needleman–Wunsch, BLOSUM62) gives:
| Reference enzyme | Characterization | % identity to PP_3157 |
|---|---|---|
| C. glutamicum HisN (Q8NS80) | First kinetic data for IMPase-type HolPase [PMID 28720084] | 36.8% (93/253) |
| M. truncatula MtHPP (G7J7Q5) | Substrate/product/by-product-bound crystal structures [PMID 26994138] | 48.8% (127/260) |
| A. thaliana IMPL2 / HISN7 (Q6NPM8) | Genetically validated; embryo-lethal null rescued by His [PMID 20023146] | 49.0% (128/261) |
All values lie well above the ~25–30% "twilight zone," so PP_3157 is a close homolog — not a marginal domain match — of enzymes for which the reaction, Mg²⁺-dependence, substrate specificity, active-site residues, 3D fold, and in-vivo His-biosynthesis role have all been directly demonstrated. This substantially strengthens confidence in the EC 3.1.3.15 / HisN assignment beyond the default "inferred from homology" level.
| Claim | Evidence type | Strength |
|---|---|---|
| EC 3.1.3.15; reaction = histidinol-P → histidinol + Pi | UniProt curated catalytic activity + TIGR02067/InterPro | High |
| Member of IMPase-like (HisN) HolPase family | InterPro IPR011809/IPR000760, Pfam PF00459, TIGRFAM TIGR02067 | High |
| Mg²⁺-dependent; active-site Asp + DPIDGT motif | UniProt metal-binding features + ortholog mutagenesis [28720084] | High |
| Specific for histidinol-P, not inositol-1-P | Biochemistry of ortholog MtHPP [26994138] | High (by homology) |
| Penultimate step of His biosynthesis (step 8/9) | UniProt pathway + reviews [26994138, 28720084] | High |
| Cytoplasmic localization | Inference from pathway + plant plastid targeting [20023146] | Moderate–High |
| In vivo HolPase role (hisN) | Genetic complementation/knockouts [20023146, 28720084, 16901339] | High (family-level) |
| Sole/dedicated HolPase; split from monofunctional IGPD | KEGG genome mapping (this study): unique K05602; hisB=K01693 (IGPD only) | High |
| Distinct from suhB/cysQ IMPase paralogs | KEGG: suhB PP_0838, cysQ PP_0261 are separate loci | High |
Principal limitation: PP_3157 itself has not been directly enzymatically or structurally characterized (UniProt PE3, inferred from homology). All mechanistic and specificity details derive from closely related, experimentally studied orthologs in the same IMPase-like HolPase family. The assignment is nonetheless robust because it rests on a specific TIGRFAM/InterPro family signature (not generic IMPase homology) and on convergent biochemical, structural, and genetic evidence from multiple orthologs.
Direct enzymatic assay of recombinant PP_3157 (kinetics on histidinol-P vs. inositol-1-P, Mg²⁺ dependence), a PP_3157 knockout to test histidine auxotrophy and possible paralog redundancy in P. putida, and an experimental or AlphaFold-validated structure to confirm the dimeric IMPase fold and active-site residues.