pydX (ordered locus name PP_4037; UniProt Q88FQ1) of Pseudomonas putida strain KT2440 encodes the electron-input (PreT-type) subunit of dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.1), an NADH-dependent iron–sulfur flavoenzyme. DPD catalyzes the committed, first step of the reductive pyrimidine catabolic pathway, reducing the pyrimidine bases uracil and thymine to their 5,6-dihydro derivatives (5,6-dihydrouracil and 5,6-dihydrothymine, respectively) at the expense of NADH. The 455-amino-acid PydX polypeptide is not the catalytic centre itself; rather, it provides the pyridine-nucleotide oxidation and intramolecular electron-relay machinery that feeds reducing equivalents to the pyrimidine-reducing active site housed on its partner subunit, PydA.
The functional enzyme is a heterotetramer of stoichiometry 2·PydA : 2·PydX (equivalent to the E. coli 2·PreA : 2·PreT architecture). PydX carries a C-terminal FAD/NAD(H)-binding Rossmann fold that oxidizes NADH and reduces its bound flavin, and an N-terminal helical ferredoxin domain harbouring tandem [4Fe-4S] clusters that shuttle electrons onward to the FMN/pyrimidine active site on the catalytic subunit PydA. This wiring—NADH → FAD (on PydX) → [4Fe-4S] clusters → FMN → pyrimidine (on PydA)—defines DPD as a member of a distinctive NADH-dependent subclass of iron–sulfur flavoenzymes. The reaction takes place in the cytoplasm.
Biologically, this pathway allows P. putida to use pyrimidines primarily as a nitrogen source: uracil/thymine are reduced by DPD, the ring is opened by dihydropyrimidinase (PydB), and the product is hydrolyzed by β-alanine synthase (HyuC), ultimately liberating NH₃ and CO₂ and yielding β-alanine. The genes are organized in a contiguous pyd cluster (PP_4034–PP_4039) that also encodes a pyrimidine transporter (PydP) and a TetR/RutR-family repressor (PydR) that controls expression. Genetic evidence (a pydA-null mutant that cannot grow on uracil or thymine as sole nitrogen source) confirms the physiological essentiality of the DPD step, and derepression in a pydR mutant confirms the regulatory logic.
| Field | Value |
|---|---|
| Gene symbol | pydX |
| Ordered locus name | PP_4037 |
| UniProt accession | Q88FQ1 |
| Length | 455 amino acids |
| Organism | Pseudomonas putida KT2440 (ATCC 47054 / DSM 6125 / NCIMB 11950) |
| EC number | 1.3.1.1 |
| Protein role | Electron-input (PreT-type) subunit of dihydropyrimidine dehydrogenase |
The gene symbol pydX correctly matches the UniProt protein description (dihydrouracil dehydrogenase, EC 1.3.1.1) and the target organism. The InterPro domains listed in the research prompt (4Fe4S_Fe-S-bd IPR017896; DPD_II IPR028261; FAD/NAD-bd_sf IPR036188; FAD/NAD-binding_dom IPR023753; Helical_ferredxn IPR009051) align exactly with the electron-input subunit of DPD. Literature on the target organism (Hidese et al. 2012) explicitly names pydX/pydA as the P. putida DPD genes, so there is no ambiguity of identity.
UniProt entry Q88FQ1 describes a 455-amino-acid protein encoded by gene pydX / ordered locus name PP_4037, annotated as "dihydrouracil dehydrogenase (NAD(+))" with EC 1.3.1.1. The catalyzed reactions correspond to Rhea RHEA:20189 (5,6-dihydrouracil + NAD⁺ = uracil + NADH + H⁺) and RHEA:28791 (5,6-dihydrothymine + NAD⁺ = thymine + NADH + H⁺). Although the reference reaction is written in the oxidative direction (as is conventional for EC 1.3.1.1), in vivo the enzyme operates in the reductive direction, consuming NADH to convert uracil → 5,6-dihydrouracil and thymine → 5,6-dihydrothymine.
The physiological requirement for this activity is demonstrated genetically: a P. putida pydA-null mutant fails to grow on minimal medium containing uracil or thymine as the sole nitrogen source (PMID: 22782928). This directly ties the DPD genes—explicitly named in that study as pydX and pydA, "tandemly arranged in the Pseudomonas putida genome"—to pyrimidine utilization. Since pydX and pydA encode the two subunits of a single enzyme, loss of the enzyme's function abolishes the committed first step and blocks pyrimidine catabolism.
"The putative DPD genes, pydX and pydA, are tandemly arranged in the Pseudomonas putida genome." — PMID: 22782928
"a pydA strain of P. putida fails to grow on a minimal media containing uracil or thymine as a sole nitrogen source, demonstrating the physiological importance of DPD in the reductive pathway" — PMID: 22782928
The domain architecture of Q88FQ1 (pydX, 455 aa) comprises: a Pyr_redox_2 FAD/NAD-binding domain (PF07992; IPR023753/IPR036188), a DPD domain II (IPR028261), a 4Fe-4S Fe-S binding domain (IPR017896; Fer4_20 / PF14691), and a helical ferredoxin domain (IPR009051). The UniProt cofactor annotation includes FMN, and the SUBUNIT annotation specifies a heterotetramer of two PreA and two PreT subunits. The partner protein, Q88FQ0 (pydA, 424 aa, PP_4038), contains the dihydroorotate-dehydrogenase-like catalytic TIM-barrel (IPR005720) plus a 4Fe-4S cluster domain (IPR017896/IPR017900)—i.e., the pyrimidine-reducing catalytic subunit.
This organization matches the biochemically characterized E. coli ortholog, which was shown to be "the first member of a novel NADH-dependent subclass of iron–sulfur flavoenzymes catalyzing the conversion of uracil to 5,6-dihydrouracil in vivo" (PMID: 21169495). In this two-subunit design, PydX supplies the electron-input chain (NADH-oxidizing flavin + iron–sulfur relay) and PydA supplies the pyrimidine-reducing active site.
"E. coli dihydropyrimidine dehydrogenase is the first member of a novel NADH-dependent subclass of iron–sulfur flavoenzymes catalyzing the conversion of uracil to 5,6-dihydrouracil in vivo." — PMID: 21169495
The gene neighborhood in P. putida KT2440 places pydX within a functionally coherent operon: PP_4035 pydP (an NCS1-family nucleoside/pyrimidine transporter, Q88FQ2), PP_4036 pydB (D-hydantoinase/dihydropyrimidinase, A0A140FWK2), PP_4037 pydX, and PP_4038 pydA, with pydR encoded nearby. Hidese et al. (PMID: 22782928) showed that pydA expression and DPD activity are elevated in a pydR mutant, establishing PydR as a repressor of the pathway; PydR is homologous to E. coli RutR. Consistent with inducible control, earlier work in Pseudomonas found that all three reductive-pathway enzymes are induced by uracil (PMID: 1903745).
The three-enzyme logic of the pathway is stated directly:
"The pathway is controlled by three enzymes: dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase and β-alanine synthase." — PMID: 22782928
"we show that PydR acts as a repressor of the pyrimidine reductive pathway in P. putida" — PMID: 22782928
"Induction of pyrimidine catabolism by uracil was observed in this pseudomonad." — PMID: 1903745
Mapping the KEGG/UniProt loci in KT2440 reveals a contiguous cluster encoding the full pathway plus transport and regulation:
| Locus | Gene | UniProt | Product / role | Pathway step |
|---|---|---|---|---|
| PP_4034 | hyuC | Q88FQ3 | N-carbamoyl-β-alanine amidohydrolase (β-alanine synthase) | Step 3 |
| PP_4035 | pydP | Q88FQ2 | NCS1 nucleoside/pyrimidine transporter | Substrate uptake |
| PP_4036 | pydB | A0A140FWK2 | D-hydantoinase / dihydropyrimidinase | Step 2 |
| PP_4037 | pydX | Q88FQ1 | DPD electron-input subunit (PreT-type) | Step 1 |
| PP_4038 | pydA | Q88FQ0 | DPD catalytic subunit (PreA-type) | Step 1 |
| PP_4039 | rutR/pydR | A0A140FWK3 | TetR/RutR-family transcriptional repressor | Regulation |
This physical co-localization of transporter, all three catabolic enzymes, and the regulator provides strong genomic-context support that pydX functions in reductive pyrimidine degradation rather than in an unrelated redox process.
InterPro/CATH mapping of Q88FQ1 (455 aa) resolves a clear two-module architecture:
At the sequence level, residues 32–65 (RQAALESARCLYCYDAPCVNACPSEIDIPSFIHR) contain the canonical CxxCxxCP ferredoxin [4Fe-4S] motif (Cys41/44/49/53), and a second cysteine cluster (Cys88/92/98/102/110) indicates a second [4Fe-4S] site. PANTHER classifies the entire protein as dihydropyrimidine dehydrogenase (PTHR43073; res 16–449). Together these features are precisely those expected of an electron-input subunit: a flavin/pyridine-nucleotide oxidase fused to an iron–sulfur electron-relay.
The structural role of these N-terminal [4Fe-4S] clusters is further illuminated by comparative work on glutamate synthase, in which four conserved N-terminal cysteines were shown to be essential not only for electron transfer but also for subunit association — a study that explicitly references "the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal β-subunit-like domain" (PMID: 15797248). This supports a model in which PydX's N-terminal ferredoxin/[4Fe-4S] module is both an electron conduit and a structural interface for heterotetramer assembly.
The InterPro domain set of pydX/Q88FQ1—{IPR028261 DPD_II; IPR023753 & IPR036188 FAD/NAD-binding; IPR009051 helical ferredoxin}—is identical to that of E. coli PreT* (P76440, b2146, 412 aa; "NAD-dependent dihydropyrimidine dehydrogenase subunit PreT"). Reciprocally, the partner pydA/Q88FQ0 domain set—{IPR017896/IPR017900 4Fe-4S; IPR013785 aldolase TIM; IPR005720 dihydroorotate DH catalytic}—matches E. coli PreA (P25889, b2147, 411 aa; cofactor [4Fe-4S]). Thus the assignment is unambiguous: pydX = PreT (electron input) and pydA = PreA (catalytic), assembling into the 2 PreA : 2 PreT* heterotetramer.
The physiological orientation of the pathway toward nitrogen scavenging is supported by growth physiology in E. coli B (PMID: 3553866), where reductive-pathway intermediates support growth roughly 14-fold better as a sole nitrogen source than as a sole carbon source:
"dihydrouracil, N-carbamoyl-beta-alanine, beta-alanine, dihydrothymine and beta-aminoisobutyric acid could sustain the growth of the bacterial cells as sole nitrogen sources by at least a fourteen-fold greater level than that observed if they were included as sole carbon sources" — PMID: 3553866
Dihydropyrimidine dehydrogenase in P. putida is a bipartite iron–sulfur flavoenzyme. PydX (PreT) is the electron-input subunit and PydA (PreA) is the catalytic subunit. In the physiologically relevant reductive direction, electrons flow from NADH through PydX's flavin and iron–sulfur relay into PydA's active site, where the pyrimidine ring is reduced across the C5–C6 double bond:
┌───────────────── PydX (PreT, electron input) ─────────────────┐
NADH → FAD/NAD Rossmann domain → [4Fe-4S] → [4Fe-4S] ────────────┐
(C-terminal, res ~142-437) (N-terminal ferredoxin, res ~6-138)│
▼
┌───────────────── PydA (PreA, catalytic) ──────────────────────┐
[4Fe-4S] → FMN → pyrimidine (uracil / thymine)
│
▼
5,6-dihydrouracil / 5,6-dihydrothymine
Assembled stoichiometry: 2 × PydA : 2 × PydX heterotetramer, operating in the cytoplasm.
DPD catalyzes step 1 of a three-enzyme reductive route that dismantles the pyrimidine ring, primarily to release nitrogen (and secondarily carbon):
uracil ──DPD (PydX/PydA)──▶ 5,6-dihydrouracil ──dihydropyrimidinase (PydB)──▶
N-carbamoyl-β-alanine ──β-alanine synthase (HyuC)──▶ β-alanine + NH3 + CO2
thymine ─DPD─▶ 5,6-dihydrothymine ─PydB─▶ N-carbamoyl-β-aminoisobutyrate
─HyuC─▶ β-aminoisobutyrate + NH3 + CO2
Substrates enter the cell via the NCS1-family transporter PydP (PP_4035), and the whole operon is held under negative control by the RutR-like repressor PydR (PP_4039), being derepressed/induced when pyrimidines are available. The ~14-fold preference for pyrimidine intermediates as nitrogen versus carbon source (PMID: 3553866) frames the biological "why": this is a nitrogen-scavenging pathway, and PydX's role is to power the committed reductive step.
The enzyme is specific for the pyrimidine bases uracil and thymine (and, correspondingly, is active on their dihydro forms in the oxidative direction). It reduces the C5=C6 double bond of the pyrimidine ring. Cytosine is not a direct substrate of this pathway (deamination to uracil is required upstream). The two documented Rhea reactions (RHEA:20189 uracil/dihydrouracil; RHEA:28791 thymine/dihydrothymine) capture this specificity.
| PMID | Title (abbrev.) | Contribution | Support / Challenge |
|---|---|---|---|
| 22782928 | Pseudomonas putida PydR represses DPD gene | Names pydX/pydA as the P. putida DPD genes; establishes PydR repressor; pydA mutant cannot use uracil/thymine as N source | Strongly supports F001, F003, F004 — the only study explicitly on the target organism/genes |
| 21169495 | E. coli DPD is a novel NAD-dependent heterotetramer | Defines the enzyme class: NADH-dependent iron–sulfur flavoenzyme heterotetramer producing 5,6-dihydrouracil | Supports F002, F006 — biochemical basis for the ortholog |
| 1903745 | Pyrimidine catabolism in P. aeruginosa | Reductive-pathway enzymes present and uracil-inducible in Pseudomonas | Supports F003 — regulation/induction |
| 3553866 | Degradation of uracil and thymine by E. coli B | Pathway intermediates support growth ~14× better as N than C source | Supports F006 — nitrogen-scavenging role |
| 15797248 | Structural role of glutamate synthase [4Fe-4S] clusters | Shows N-terminal β-subunit [4Fe-4S] clusters are structural and reference the 3D structure of DPD as a model | Contextual support for F005 — role of N-terminal [4Fe-4S]/ferredoxin module in a homologous enzyme |
The evidence is a combination of: (i) direct genetic evidence in the target organism (the pydA growth phenotype and pydR derepression, PMID: 22782928); (ii) biochemical characterization of the close E. coli ortholog (PMID: 21169495); (iii) bioinformatic/structural inference from InterPro/CATH domain mapping and conserved cysteine motifs (F005, F006); and (iv) physiological/comparative context (PMID: 1903745, PMID: 3553866).
The gene symbol pydX is correctly matched to the target: it is the P. putida KT2440 gene (PP_4037, UniProt Q88FQ1) for the electron-input (PreT-type) subunit of dihydropyrimidine dehydrogenase (EC 1.3.1.1). All lines of evidence—organism-specific genetics, ortholog biochemistry, genomic context, and domain/structural bioinformatics—converge on a single, coherent function: PydX supplies NADH-derived reducing equivalents, via an FAD/NAD Rossmann domain and tandem [4Fe-4S] clusters, to the catalytic subunit PydA, enabling the cytoplasmic reduction of uracil and thymine as the committed first step of the reductive pyrimidine catabolic pathway that P. putida uses chiefly to acquire nitrogen.