PP_0375 (Q88QV9): A Family-S9 Serine Peptidase of the Pseudomonas putida KT2440 Pyrroloquinoline-Quinone (PQQ) Biosynthesis Operon

Target: PP_0375 · UniProt: Q88QV9 · Organism: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / KT2440), taxid 160488
Protein length: 612 aa · Gene synonyms: pqqG (operon terminal gene); ortholog of P. aeruginosa pqqH (PA1990)

Summary

PP_0375 encodes a family-S9 (prolyl oligopeptidase clan SC) serine peptidase — a catalytically competent α/β-hydrolase whose primary molecular function is peptide-bond hydrolysis. The gene sits as the terminal ("G") gene of the pyrroloquinoline-quinone (PQQ) biosynthesis operon of P. putida KT2440, whose gene order has been mapped by RT-PCR as pqqFABCDEG (loci PP_0375–PP_0381). PP_0375 is the ~53 %-identical ortholog of P. aeruginosa pqqH/PA1990, the peptidase gene cotranscribed with pqqABCDE. Its role therefore lies within PQQ cofactor maturation, a ribosomally-synthesized-and-post-translationally-modified-peptide (RiPP) pathway that builds the redox cofactor PQQ from a glutamate and a tyrosine embedded in the 23-residue precursor peptide PqqA.

Structurally, PP_0375 has the canonical two-domain prolyl-oligopeptidase architecture: an N-terminal seven-bladed β-propeller domain that gates the active site, packed against a C-terminal α/β-hydrolase catalytic domain carrying the Peptidase_S9 module (Pfam PF00326). The catalytic Ser469–His582–Asp550 triad is geometrically intact in the high-confidence AlphaFold model (global pLDDT 93.8; triad distances 3.0 Å and 2.78 Å), with Ser469 sitting in a classic G-x-S-x-G "nucleophile elbow." Because the β-propeller covers the active site, this class of enzyme is intrinsically selective for short peptides (oligopeptidase behavior) rather than folded proteins — the enzyme sees a buried, gated pocket rather than an open surface groove.

Functionally, the PQQ operon of P. putida carries two proteases of different mechanistic clans, doing two different jobs. The essential core excision of the PqqE-cross-linked Glu–Tyr di-amino-acid from PqqA is performed by PqqF (PP_0381), an M16 zinc "inverzincin" whose deletion abolishes PQQ synthesis. In contrast, deletion of the S9 peptidase (pqqH, the PP_0375 ortholog) in P. aeruginosa does not block PQQ synthesis but abolishes PQQ excretion into the culture supernatant. PP_0375 therefore functions in the cytoplasm during cofactor maturation/release, and the PQQ it helps produce is exported to the periplasm, where it serves as the redox cofactor of quinoprotein dehydrogenases (glucose dehydrogenase Gcd, and the alcohol dehydrogenases PedE/PedH), driving glucose→gluconate oxidation (mineral-phosphate solubilisation) and alcohol/aldehyde oxidation. The exact peptide substrate of PP_0375 has not been experimentally established and remains the principal knowledge gap.

Gene/Protein Identity Verification

Before presenting findings, the mandatory identity checks were satisfied:

Conclusion: The identity is secure. PP_0375 is the operonic S9 peptidase (pqqG) of the P. putida KT2440 PQQ cluster.

Key Findings

Finding 1 — PP_0375 is a Peptidase S9 serine hydrolase with an intact Ser–His–Asp catalytic triad

UniProt Q88QV9 (612 aa) carries a consistent, mutually corroborating stack of domain annotations that place it firmly in the prolyl oligopeptidase family (clan SC, family S9): Pfam PF00326 (Peptidase_S9), InterPro IPR001375 (Peptidase_S9_cat), IPR029058 (α/β-hydrolase fold), IPR050585, PANTHER PTHR43056:SF5, eggNOG COG1506 (dipeptidyl-/acylamino-acid peptidase), and Gene3D 3.40.50.1820 (the α/β-hydrolase superfamily fold).

The catalytic machinery is not merely annotated but structurally verified. The nucleophile Ser469 lies in a canonical G-x-S-x-G elbow (sequence …RGGSAGG…), the signature "nucleophile elbow" of α/β-hydrolases. In the AlphaFold model AF-Q88QV9 (global pLDDT 93.8, i.e. very high confidence), the triad is geometrically complete and correctly oriented for catalysis:

• Ser469 Oγ – His582 Nε2 = 3.0 Å (hydrogen-bond distance for the charge-relay proton transfer)
• His582 Nδ1 – Asp550 Oδ2 = 2.78 Å (the second charge-relay contact)
• All three triad residues have local pLDDT > 86 (individually reliable)

The InterPro-derived Gene Ontology terms are serine-type peptidase activity (GO:0008236) and proteolysis (GO:0006508). Together these establish, with high confidence, that PP_0375 is a functional serine peptidase and not a degenerate/pseudo-enzyme.

Finding 2 — PP_0375 is the P. putida ortholog of pqqH, a peptidase embedded in the PQQ biosynthesis operon

Genomic context is the decisive evidence. In the STRING network (organism 160488), PP_0375's highest-confidence functional partners are the PQQ-biosynthesis genes, and the supporting evidence channel is genomic neighborhood (nscore) — meaning these genes are co-located and co-inherited:

PP_0375 sits immediately adjacent to pqqE (PP_0376) — precisely the operonic position occupied by pqqH in P. aeruginosa. A Needleman–Wunsch global alignment shows PP_0375 (612 aa) is 52.9 % identical (344 identities over 650 aligned columns) to P. aeruginosa pqqH/PA1990 (Q9I2B9, 608 aa). This level of identity across nearly the full length indicates clear orthology, not merely shared domain content.

The functional identity of pqqH was established experimentally in P. aeruginosa (PMID: 19902179):

"Gene PA1990 of Pseudomonas aeruginosa, located downstream of pqqE and encoding a putative peptidase, was shown to be involved in excretion of PQQ into the culture supernatant. This gene is cotranscribed with the pqqABCDE cluster and was named pqqH."

PP_0375 is the P. putida ortholog occupying the same operonic position.

Finding 3 — PP_0375 (pqqG/pqqH) functions in PQQ cofactor biosynthesis for periplasmic quinoprotein glucose dehydrogenase

RT-PCR mapping of the P. putida KT2440 cluster defines its gene order as pqqFABCDEG (An & Moe 2016, PMID: 27287323). PP_0375 is the terminal "G" gene immediately after pqqE (PP_0376) — i.e. the peptidase-encoding gene, ortholog of P. aeruginosa pqqH/PA1990.

The biological purpose of the operon is to synthesize PQQ, the redox coenzyme of the periplasmic glucose dehydrogenase (Gcd) that oxidizes glucose to gluconic acid, enabling mineral-phosphate solubilization in the rhizosphere:

"Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme." — PMID: 27287323

"The pqq gene cluster (pqqFABCDEG) encodes at least two independent transcripts" — PMID: 27287323

Biochemically, PQQ is a RiPP built from a Glu and a Tyr embedded in the ribosomal precursor peptide PqqA. The precursor is cross-linked (C–C bond between the Glu Cγ and Tyr Cε) by the radical-SAM enzyme PqqE (assisted by the RRE chaperone PqqD), after which a protease/peptidase must excise the modified di-amino-acid before PqqB (dual hydroxylase) and PqqC (eight-electron oxidase/cyclase) complete the cofactor:

"The PqqA peptide is recognised by PqqE, which links the C9 and C9a, afterwards it is accepted by PqqF which cuts out the linked amino acids." — PMID: 18371220

Finding 4 — Two operon proteases, two roles: PqqF (M16) excises PqqA; PP_0375 (pqqG/pqqH, S9) is linked to PQQ excretion

Neighbor annotations (UniProt, organism 160488) confirm the complete operon and, critically, that it encodes two peptidases of entirely different catalytic clans:

Crystallography shows PqqF has a clamshell inverzincin fold with a large (~9,400 ų) internal chamber proposed to bind PqqA and excise the PqqE-cross-linked Glu–Tyr; pqqF deletion abolishes PQQ biosynthesis (Wei et al. 2016):

"After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF." — PMID: 27231346

"we demonstrated that the pqqF gene is essential for PQQ biosynthesis" — PMID: 27231346

By contrast, deletion of the S9 peptidase pqqH in P. aeruginosa does not block PQQ synthesis but abolishes PQQ excretion into the supernatant (PMID: 19902179). This establishes a division of labor: PqqF performs the essential core excision step, while PP_0375/pqqG/pqqH plays a distinct, non-essential-for-synthesis role tied to release/excretion of mature PQQ. The precise molecular (peptide) substrate of PP_0375 has not been experimentally determined.

Finding 5 — Two-domain prolyl-oligopeptidase architecture implies short-peptide (oligopeptidase) selectivity

InterPro/SUPFAM/Gene3D domain mapping of Q88QV9 reveals the classic two-domain organization of the prolyl-oligopeptidase family:

• N-terminal β-propeller ("DPP6 N-terminal domain-like," SCOP SSF82171, residues ~44–337)
• C-terminal α/β-hydrolase catalytic domain (Gene3D G3DSA:3.40.50.1820, res ~356–612; SUPFAM SSF53474, res ~358–603), carrying the Peptidase_S9 module (PF00326 / IPR001375, res ~401–607) with the Ser469–Asp550–His582 triad
• PANTHER PTHR43056 (Xaa-Pro dipeptidyl-peptidase / cocaine esterase) spans res 4–608

In the AlphaFold model (pLDDT 93.8) the two domains pack tightly (75 Cα–Cα inter-domain contacts < 8 Å; centroid separation 32.5 Å), and the catalytic Ser469 lies in an enclosed pocket (165 protein heavy atoms within 10 Å of Ser469 Oγ). This is a covered/gated active site, not an open surface groove.

The functional implication is well established for this fold family: the β-propeller acts as a molecular sieve/gate that admits only small, unstructured peptides to the buried catalytic center, excluding large folded proteins. PP_0375 is therefore predicted to be an oligopeptidase — selective for short peptides, which is mechanistically consistent with acting on the small PqqA-derived/PQQ-maturation intermediates rather than degrading bulk protein.

Finding 6 — The PQQ cofactor served by PP_0375 feeds multiple periplasmic quinoprotein dehydrogenases

UniProt (organism 160488) confirms three PQQ-dependent apo-dehydrogenases in KT2440 that consume the cofactor whose biosynthesis PP_0375 supports:

Wehrmann et al. 2017 (PMID: 28655819) purified and characterized PedE/PedH as functionally redundant periplasmic PQQ-ADHs for oxidation of alcohols and aldehydes (relevant to volatile-organic-compound detoxification and catabolism); PedH was the first lanthanide-dependent PQQ-ADH described in a non-methylotroph:

"many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant" — PMID: 28655819

This closes the loop: the cofactor PP_0375 helps mature is exported and used by periplasmic quinoproteins that drive both glucose→gluconate oxidation (Gcd; phosphate solubilisation, environmental acidification) and alcohol/aldehyde oxidation (PedE/PedH).

Mechanistic Model / Interpretation

The PQQ biosynthesis operon and PP_0375's place in it

   P. putida KT2440 pqq operon  (order: pqqFABCDEG)

   PP_0381   PP_0380  PP_0379  PP_0378  PP_0377  PP_0376   PP_0375
   ┌─────┐   ┌────┐   ┌────┐   ┌────┐   ┌────┐   ┌────┐    ┌───────┐
   │ pqqF│   │pqqA│   │pqqB│   │pqqC│   │pqqD│   │pqqE│    │ pqqG  │  ← TARGET
   │ M16 │   │23aa│   │hydr│   │cycl│   │RRE │   │rSAM│    │  S9   │
   │prot.│   │prec│   │oxyl│   │oxid│   │chap│   │cycl│    │peptid.│
   └─────┘   └────┘   └────┘   └────┘   └────┘   └────┘    └───────┘
   ESSENTIAL                                                 EXCRETION-
   for PQQ                                                   linked
   synthesis

Biosynthetic route (RiPP pathway) and the two-protease division of labor

   Ribosome
      │
      ▼
   PqqA (23-aa precursor, contains Glu + Tyr)
      │   bound by PqqD (RRE chaperone)  ── presents to ──►  PqqE (radical-SAM)
      ▼
   PqqA with Glu-Cγ ── C–C ── Tyr-Cε  cross-link
      │
      │  ┌── CORE EXCISION ──────────────────────────────┐
      ▼  │  PqqF (M16 inverzincin, PP_0381) cleaves the   │
   excised │  cross-linked di-amino-acid from PqqA.       │  ESSENTIAL
   Glu–Tyr │  Δpqqf → NO PQQ.                             │
           └──────────────────────────────────────────────┘
      │
      ▼   PqqB (hydroxylation) + PqqC (ring closure, 8e⁻/8H⁺ oxidation)
      ▼
   ★ PQQ (mature cofactor) ★
      │
      │  ┌── EXCRETION / RELEASE ─────────────────────────┐
      │  │  pqqG/pqqH (S9 serine peptidase, PP_0375)      │  NON-ESSENTIAL
      │  │  ΔpqqH → PQQ still made but NOT excreted        │  for synthesis;
      │  └────────────────────────────────────────────────┘  needed for export
      ▼
   PERIPLASM  ─►  Gcd (glucose→gluconate), PedE/PedH (alcohol/aldehyde ox.)

Synthesis

PP_0375 is best understood as a cytoplasmic, short-peptide-selective serine oligopeptidase acting in PQQ cofactor maturation, distinct in both clan (S9 serine vs. M16 zinc) and phenotype (excretion vs. essential synthesis) from its operon partner PqqF. The structural evidence (intact triad, gated β-propeller active site, oligopeptidase architecture) is fully consistent with a role hydrolyzing a small peptide substrate during the late steps of PQQ maturation or release. The strongest experimental anchor for its physiological role is the P. aeruginosa pqqH knockout phenotype — loss of PQQ excretion — transferred by ~53 % orthology to PP_0375. Its exact substrate (whether a residual PqqA-derived peptide, a maturation intermediate, or a distinct target coupled to export) remains undetermined.

Evidence Base

Consistency / tension: The literature is internally consistent that PQQ biosynthesis requires a protease for di-amino-acid excision, and that different organisms deploy different peptidases. A key nuance (PMID: 31427437) is that the maturation protease is a "missing piece" that varies across taxa — in some α-proteobacteria it is a two-component heterodimer, in Klebsiella/Methylobacterium it is PqqF (M16), and P. aeruginosa/P. putida additionally carry the S9 pqqH/pqqG. The evidence assigns the essential excision to PqqF in Pseudomonas and an excretion-linked role to PP_0375/pqqG — but does not exclude an accessory maturation role for the S9 enzyme.

Limitations and Knowledge Gaps

1. No direct experimental characterization of PP_0375 itself. All functional inference derives from (a) orthology to P. aeruginosa pqqH (~53 % identity), (b) genomic operon context, and (c) structural prediction. No P. putida PP_0375 knockout, purified enzyme assay, or crystal structure exists in the reviewed literature.
2. The molecular substrate is undefined. Whether PP_0375 cleaves a residual PqqA fragment, a PQQ-maturation intermediate, or an unrelated peptide coupled to export is not established. The "excretion" phenotype of pqqH could reflect a direct role in export machinery processing rather than PQQ chemistry.
3. The AlphaFold-based active-site and oligopeptidase-selectivity claims are predictions, not experimental structures — high-confidence (pLDDT 93.8) but unvalidated by crystallography or mutagenesis.
4. Localization is inferred. A cytoplasmic role during maturation is the parsimonious interpretation, but the excretion phenotype raises the possibility of association with the inner membrane or export apparatus; no experimental subcellular-localization data were found.
5. Operon transcription nuance: the KT2440 cluster produces "at least two independent transcripts," so pqqG expression may be regulated separately from the core pqqABCDE, but this was not investigated in detail here.

Proposed Follow-up Experiments / Actions

1. Targeted gene deletion (ΔPP_0375) in P. putida KT2440, measuring (i) intracellular vs. extracellular PQQ (LC-MS or the classic E. coli GDH-reconstitution bioassay), (ii) glucose→gluconate flux, and (iii) periplasmic Gcd/PedE-PedH activity. This directly tests whether the pqqH "excretion" phenotype transfers to PP_0375.
2. Recombinant expression and enzymology. Purify PP_0375 and assay peptidase activity against candidate substrates: synthetic PqqA-derived peptides, cross-linked Glu–Tyr intermediates, and generic oligopeptide libraries (e.g., internally quenched fluorogenic peptides) to define the length and sequence specificity predicted by the gated β-propeller.
3. Active-site mutagenesis. Ser469Ala (and His582Ala) catalytic-dead variants to confirm the triad is required for the physiological phenotype, decoupling catalytic vs. possible scaffolding roles.
4. Structural validation. Solve a crystal or cryo-EM structure of PP_0375 to confirm the β-propeller / α-β-hydrolase two-domain arrangement and the buried active site, and to visualize the substrate-access channel.
5. Interaction / localization mapping. Co-purification or BioID with operon partners (PqqF, PqqE, PqqB/C) and with export components; cell-fractionation to test cytoplasmic vs. membrane-associated localization.
6. Comparative genomics of the two-protease pattern. Systematically map which PQQ-producing genomes carry PqqF, pqqG/pqqH, both, or the α-proteobacterial two-component protease, to clarify the evolutionary logic of redundancy and the specific selective role of the S9 enzyme.

Report generated from a 5-iteration autonomous investigation: 6 confirmed findings, 15 papers reviewed. All functional claims are supported by the cited literature; claims resting on structure prediction or orthology are flagged as such.

Artifacts

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• OpenScientist final report

Citations

1. PMID:19902179
2. PMID:27287323
3. PMID:18371220
4. PMID:27231346
5. PMID:28655819
6. PMID:31427437