Gene: purF (ordered locus PP_2000) · UniProt: Q88LD5 · Enzyme: Amidophosphoribosyltransferase / glutamine phosphoribosylpyrophosphate amidotransferase (GPATase, ATase) · EC 2.4.2.14 · Organism: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / KT2440)
purF encodes amidophosphoribosyltransferase (GPATase), the enzyme that catalyzes the first committed and rate-controlling step of de novo purine nucleotide biosynthesis. In the cytoplasm it transfers the amide nitrogen of L-glutamine to 5-phospho-α-D-ribose-1-diphosphate (PRPP), producing 5-phospho-β-D-ribosylamine (PRA), L-glutamate, and pyrophosphate. It is a two-domain glutamine amidotransferase: an N-terminal Ntn-hydrolase glutaminase domain (catalytic Cys1) generates ammonia, which is channeled through an internal tunnel to a C-terminal phosphoribosyltransferase (PRT) domain that attaches it to PRPP. Its activity is gated by ordered substrate binding (PRPP first, which activates the glutaminase site) and feedback-inhibited by purine nucleotides (AMP/GMP/IMP), while the gene is transcriptionally repressed by the purine-responsive PurR repressor. The product PRA feeds both the purine pathway (→ IMP → AMP/GMP) and thiamine biosynthesis.
Gene-identity verification: Confirmed. The gene symbol, EC number, InterPro domains (GATase_2, Ntn-hydrolase N, PRTase), organism, and the direct Q88LD5 sequence (501 aa beginning M-C-G-I-V-G, the canonical GPATase N-terminus; 65% identical to experimentally characterized E. coli PurF) all agree. This is a genuine, well-characterized amidophosphoribosyltransferase, not an ambiguous or mis-annotated symbol.
Reaction (EC 2.4.2.14):
PRPP + L-glutamine + H₂O → 5-phospho-β-D-ribosylamine (PRA) + L-glutamate + PPᵢ
This is the entry reaction of de novo purine biosynthesis. In Salmonella/E. coli genetics, "Glutamine phosphoribosyl pyrophosphate (PRPP) amidotransferase is the product of the purF gene … and catalyzes the synthesis of PRA from PRPP and glutamine. Strains lacking PurF require exogenous addition of purines for growth" (Koenigsknecht et al., 2012, PMID 23133571). The enzyme is also confirmed as a distinct, competitively inhibitable target: feruloyl amide "is a competitive inhibitor of glutamine PRPP amidotransferase (PurF)" (Pisithkul et al., 2015, PMID 26070680).
Substrate specificity and kinetics. The two substrates are the phosphoribosyl donor PRPP and the amide-nitrogen donor L-glutamine (free ammonia can substitute at high concentration in vitro, but glutamine is the physiological donor). Kinetic analysis of the mechanistically conserved enzyme gave dissociation constants of 80 µM for PRPP and 420 µM for L-glutamine, "with P-Rib-PP bound first with positive cooperativity for interaction with a second site on the catalytically active dimer" (Schoettle et al., 1997, PMID 9174353). Thus catalysis follows an ordered mechanism in which PRPP binds before glutamine.
GPATase is a bifunctional, two-domain enzyme:
Active-site chemistry (from E. coli crystallography + mutagenesis, Kim et al., 1996, PMID 8663035): the free α-amino group of Cys1 acts as the proton acceptor/donor; Asn101 and the Gly102 backbone form the oxyanion hole; Arg73 and Asp127 bind glutamine; and Tyr74 couples the glutamine and PRPP sites.
Substrate-gated activation. The basal PRPP-independent glutaminase activity is only ~0.3% of the fully active enzyme; "Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine" (PMID 8663035). The ring oxygen of PRPP is required for this activating conformational change (Kim et al., 1995, PMID 7542237). This structural gating ensures glutamine is not wastefully hydrolyzed unless the acceptor PRPP is present — the mechanistic counterpart of the PRPP-first ordered kinetics.
Intramolecular ammonia channeling. The ammonia generated at the glutaminase site is not released to solvent; GPATase "catalyzes the synthesis of 5′-phosphoribosylamine in a reaction that involves the translocation of ammonia along an intramolecular tunnel linking the two active sites," behaving as a "pipe" through which ammonia travels; the L415A mutation reduces channel efficiency (Wang et al., 2009, PMID 19921932).
GPATase is a soluble cytoplasmic enzyme. All structurally characterized bacterial orthologs (E. coli, B. subtilis) are cytosolic proteins crystallized as soluble oligomers (PMID 9514258; 8663035), and the P. putida sequence has no signal peptide or transmembrane segment. It therefore carries out its function in the cytoplasm, where PRPP and glutamine are supplied by central metabolism. (The mammalian cytosolic "purinosome" metabolon is a eukaryote-specific organization and is not expected in bacteria; notably, even in humans amidophosphoribosyltransferase is the one DNPB enzyme that does not stably join the PAICS-centered purinosome interactions — He et al., 2022, PMID 35331738.)
PurF initiates the ten-step de novo purine pathway: purF → purD → purN/purT → purL → purM → purK/purE → purC → purB → purH, yielding IMP, from which AMP and GMP are made. Its product PRA is also the branch-point precursor of thiamine (vitamin B1) biosynthesis: "Phosphoribosylamine (PRA) is an intermediate in the biosynthetic pathway that is common to thiamine and purines" (PMID 23133571). By consuming PRPP and glutamine, PurF sits at the intersection of nucleotide supply, one-carbon/nitrogen metabolism, and central carbon metabolism.
PurF flux is controlled at two layers:
Bacterial GPATases form two subfamilies: "The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes … a common ancestor … may have included an Fe-S cluster" (PMID 9514258). The B. subtilis [4Fe-4S] cluster is remote from the active sites and confers oxygen-triggered turnover rather than catalysis. Q88LD5 is a gammaproteobacterial, E. coli-like, metal-free GPATase (HAMAP rule MF_01931), so it is not expected to be intrinsically O₂-sensitive via a metal center — consistent with P. putida's aerobic lifestyle.
Direct evidence for the P. putida protein: Q88LD5 is 501 aa, begins MCGIVGIVG (conserved Cys1 nucleophile motif), and is 65.1% identical to E. coli PurF (P0AG16) over the full length (Needleman-Wunsch, this work). Crucially, all six experimentally-defined catalytic residues are perfectly conserved in Q88LD5 by alignment (this work): the nucleophile Cys1, glutamine-binding Arg73 and Asp127, inter-site coupling Tyr74, and oxyanion-hole Asn101/Gly102 (residues defined in E. coli by Kim et al., PMID 8663035). This residue-level conservation justifies transferring the E. coli experimental mechanism directly to the P. putida ortholog.
Supported:
- H1: purF encodes amidophosphoribosyltransferase (EC 2.4.2.14) catalyzing PRPP + Gln → PRA. ✔ (PMIDs 23133571, 26070680; sequence)
- H2: Two-domain Ntn-hydrolase mechanism with Cys1 nucleophile and PRPP-gated glutaminase. ✔ (PMIDs 8663035, 10049369, 7542237)
- H3: Intramolecular ammonia channeling between active sites. ✔ (PMID 19921932)
- H4: Ordered kinetics (PRPP first); feedback inhibition by AMP/GMP/IMP. ✔ (PMIDs 9174353, 9514258)
- H5: Dual regulation — allosteric + PurR transcriptional repression. ✔ (PMIDs 8089849, 14741201, 7638212)
- H6: Cytoplasmic localization; entry to purine + thiamine pathways. ✔ (PMIDs 23133571, 9514258)
- H7: P. putida enzyme is a metal-free, E. coli-type GPATase. ✔ (PMID 9514258; 65% identity)
Refuted / not applicable:
- The bacterial enzyme does not organize into a mammalian-style purinosome, and (unlike the B. subtilis enzyme) is not predicted to carry an oxygen-sensitive Fe-S cluster.
Key references: PMID 23133571, 26070680, 8663035, 10049369, 7542237, 19921932, 9174353, 9514258, 8089849, 14741201, 7638212, 35331738. Sequence analyses (Q88LD5 vs P0AG16) performed in this study.