Gene: ilvE | Ordered locus: PP_3511 | UniProt: Q88H54 (Q88H54_PSEPK) | EC: 2.6.1.42 | Length: 339 aa
Organism: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950 / KT2440)
The gene ilvE (ordered locus PP_3511; UniProt Q88H54) of Pseudomonas putida KT2440 encodes a branched-chain-amino-acid aminotransferase (BCAT), historically known as "transaminase B," classified as EC 2.6.1.42. This is a soluble, cytoplasmic, pyridoxal-5′-phosphate (PLP)-dependent enzyme of the class-IV / fold-type IV aminotransferase family. The family assignment is fully consistent with the gene symbol, the enzyme's InterPro domain architecture (Aminotrans_IV IPR001544, Aminotrans_IV_CS IPR018300, B_amino_transII IPR005786, BCAT-like_C IPR043132), and a ~48% amino-acid identity to the biochemically characterized Escherichia coli IlvE ortholog. Target identity was verified against the UniProt reference; the gene symbol, organism, protein family, and domain content all converge on a single, unambiguous functional annotation, and no evidence of gene-symbol ambiguity was encountered.
Functionally, IlvE catalyzes the reversible transamination that interconverts L-glutamate/2-oxoglutarate (α-ketoglutarate) with the three branched-chain 2-oxo acids and their corresponding amino acids — valine, isoleucine, and leucine. In the biosynthetic direction, IlvE performs the final step of branched-chain amino acid (BCAA) biosynthesis, aminating the 2-oxo acids produced by the upstream acetohydroxyacid pathway (ilvBN/ilvC/ilvD) and the leucine pathway (leuABCD). In the catabolic direction, the same enzyme performs the first committed step of BCAA degradation, removing the α-amino group to regenerate the branched-chain 2-oxo acids that are the direct substrates of the inducible branched-chain 2-oxo (keto) acid dehydrogenase (Bkd) complex encoded by the bkd operon. IlvE thus sits at the central transamination node coupling BCAA anabolism and catabolism to cellular glutamate/nitrogen metabolism.
Mechanistically, the enzyme operates by a ping-pong bi-bi kinetic mechanism through a PLP↔PMP (pyridoxamine-5′-phosphate) cofactor cycle, with catalysis occurring on the distinctive re-face of the cofactor — a hallmark of fold-type IV enzymes. The catalytic Schiff-base lysine that forms the internal aldimine with PLP is strictly conserved in Q88H54 (Lys183, within the invariant A-A-K-x-G-G-N-Y motif). Substrate and stereospecificity for the L-branched-chain substrates are set by a hydrophobic active-site pocket and glutamate-selective polar contacts, features established by crystallographic studies of homologous BCATs and structurally conserved in bacterial IlvE. The E. coli-type BCAT (closest characterized ortholog) is a homohexamer arranged as a double trimer, distinguishing it from the homodimeric BCATs of most other organisms.
The UniProt annotation, gene symbol, organism, and domain architecture are mutually consistent and unambiguous:
There is no evidence of gene-symbol ambiguity: the ilvE symbol, EC number, family, and domains all converge on the same enzyme, and the closest experimentally characterized homolog (E. coli transaminase B) has exactly this activity.
UniProt Q88H54 annotates PP_3511/ilvE as a Branched-chain-amino-acid aminotransferase, EC 2.6.1.42, a class-IV PLP-dependent enzyme (Aminotrans_IV, IPR001544). BCATs catalyze the reversible transamination between L-glutamate and the branched-chain 2-oxo acids: 2-oxoisovalerate ↔ valine, (S)-3-methyl-2-oxopentanoate ↔ isoleucine, and 4-methyl-2-oxopentanoate ↔ leucine. In the biosynthetic direction IlvE catalyzes the last (transamination) step; because the reaction is freely reversible, the same enzyme initiates BCAA catabolism.
The functional assignment is anchored by direct study of orthologs. In Mycobacterium tuberculosis, the IlvE enzyme "is responsible for the final step of the synthesis of the branched-chain amino acids isoleucine, leucine, and valine" (PMID: 27780341). Classical genetics of the ilv operon named ilvE "transaminase B," identifying it among the "structural genes ilvE (transaminase B), ilvD (dehydrase), and ilvA (threonine deaminase)" (PMID: 14273641). The substrate scope of the enzyme class is defined by the observation that "Branched chain aminotransferases (BCATs) catalyze transamination of the branched chain amino acids (BCAAs) leucine, isoleucine, and valine" (PMID: 11642362).
The Q88H54 domain architecture (Aminotrans_IV, Aminotrans_IV_CS, B_amino_transII, BCAT-like_C) places the enzyme firmly in the class-IV / fold-type IV PLP enzyme family. As the authoritative BCAT review states, "Structurally, the BCATs belong to the fold type IV class of pyridoxal phosphate (PLP) enzymes" (PMID: 11642362). The family is mechanistically distinctive: "Catalysis is on the re face of the PLP cofactor, whereas in other classes, catalysis occurs from the si face of PLP" (PMID: 11642362). This distinguishes IlvE from the aspartate-aminotransferase (fold-type I) superfamily. The fold-type IV family also includes D-amino-acid aminotransferase and 4-amino-4-deoxychorismate lyase, which share the same active-site architecture around the cofactor (PMID: 10876155).
Kinetically, the M. tuberculosis IlvE study confirmed that "As seen in other aminotransferases, MtIlvE displays a ping-pong kinetic mechanism" (PMID: 27780341). This is consistent with the two half-reactions of transamination proceeding through a PLP↔PMP cofactor cycle mediated by an active-site lysine Schiff base: the amino donor binds first, transfers its amino group to PLP (forming PMP) and departs as a 2-oxo acid, after which the acceptor 2-oxo acid binds and is aminated.
Bacteria characteristically use one BCAT for both anabolic and catabolic transamination: "The BCATs are widely distributed in the bacterial kingdom, where they are involved in the synthesis/degradation of the BCAAs. Bacteria contain a single BCAT" (PMID: 11642362). Bacterial BCATs are soluble cytoplasmic enzymes, in contrast to the eukaryotic mitochondrial BCATm isozyme. Q88H54 carries no predicted signal peptide and no transmembrane segment, consistent with a cytosolic protein that is not membrane-associated.
Positionally, in P. putida KT2440 the enzyme sits at the branch point of BCAA anabolism (downstream of the ilvBN/ilvC/ilvD acetohydroxyacid pathway and the leuABCD leucine pathway) and BCAA catabolism (upstream of the branched-chain 2-oxo acid dehydrogenase, Bkd). In both directions it uses glutamate/2-oxoglutarate as the nitrogen carrier, coupling BCAA carbon skeletons to the cell's central nitrogen pool.
The definitive substrate-specificity data come from direct enzymology of the E. coli ilvE product (Transaminase B, EC 2.6.1.42), the closest well-characterized ortholog. Purified enzyme "catalyzed transamination between alpha-ketoglutarate and l-isoleucine, l-leucine, l-valine, and, to a lesser extent, l-phenylalanine and l-tyrosine, the latter reacting very sluggishly," and was "free of aspartate transaminase and of transaminase C" with an "absence of a separate valine-alpha-ketoglutarate activity" — i.e., a single enzyme handles all three BCAAs (PMID: 378964). The apparent Km values reveal a preference for the 2-oxo-acid substrates: "The apparent K(m) values for the branched-chain alpha-ketoacids were smaller than those for the corresponding amino acids" (PMID: 378964); the lowest Km was for the isoleucine 2-oxo acid, the highest for L-valine.
The physiological dominance of IlvE — and its partial redundancy for some substrates — is quantified by a Lactococcus lactis ilvE knockout, in which "the mutant retained <2, 4.5, 43, 40, and 76% of its aminotransferase activity with Ile, Val, Leu, Met, and Phe, respectively" (PMID: 10831406). This establishes IlvE as the principal (near-sole) aminotransferase for isoleucine and valine, while leucine, methionine, and phenylalanine transamination is partly served by other enzymes.
| Substrate | Relative activity retained in ilvE knockout (L. lactis) | Interpretation |
|---|---|---|
| Isoleucine | < 2% | IlvE essentially the sole transaminase |
| Valine | 4.5% | IlvE strongly dominant |
| Leucine | 43% | IlvE major, partial redundancy |
| Methionine | 40% | Partial contribution |
| Phenylalanine | 76% | Minor contribution; largely redundant |
Analysis of the E. coli Transaminase B established that "the oligomeric structure of the enzyme, as determined by analytical ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was confirmed to be that of a hexamer," with a molecular weight of ~182,000 and apparently identical subunits "consistent with a double-trimer arrangement" (PMID: 378964). This quaternary structure is unusual within the family: "Except for the Escherichia coli and Salmonella proteins, which are homohexamers arranged as a double trimer, the BCATs are homodimers" (PMID: 11642362). Because P. putida IlvE is a close E. coli-type ortholog, a hexameric assembly is the most likely quaternary state, though this has not been directly demonstrated for Q88H54.
For subcellular localization, the Lactococcus IlvE study reported that "Hydrophobicity plot analysis of the deduced amino acid sequence and the lack of a signal peptide sequence suggest IlvE is a cytosolic protein" (PMID: 10831406). Q88H54 likewise has no predicted signal peptide or transmembrane region, supporting cytoplasmic localization.
P. putida degrades branched-chain amino acids via the inducible branched-chain keto acid dehydrogenase (Bkd) multienzyme complex, encoded by the bkd operon. Transcription is controlled by the activator BkdR, and "the L-branched-chain amino acids and D-leucine are the inducers of the bkd operon" (PMID: 10217783). The operon is additionally subject to catabolite repression: "The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression," mediated by Crc (PMID: 10648542).
In the catabolic direction, IlvE removes the α-amino group from valine/isoleucine/leucine to yield the corresponding branched-chain 2-oxo acids, which are the direct substrates oxidatively decarboxylated by the Bkd complex. IlvE thereby supplies the entry substrates for the bkd pathway while regenerating glutamate from 2-oxoglutarate. In the biosynthetic direction, IlvE acts downstream of the ilvBN/ilvC/ilvD (valine/isoleucine) and leuABCD (leucine) pathways to aminate the same 2-oxo acids. The regulatory apparatus of the catabolic branch is well characterized in P. putida: BkdR is a tetrameric Lrp homolog that binds the bkdR–bkdA1 intergenic region (PMID: 7836297, PMID: 8670279, PMID: 9068646), with catabolite repression in rich medium acting posttranscriptionally on bkdR expression (PMID: 10648543).
A Needleman–Wunsch global alignment of the UniProt sequences shows that Q88H54 (339 aa) shares 47.7% amino-acid identity (132/277 aligned positions) with E. coli IlvE/transaminase B (P0AB80, 309 aa), the enzyme whose EC 2.6.1.42 activity and substrate specificity were directly measured (PMID: 378964). For a single-domain fold-type IV aminotransferase, ~48% identity is comfortably within the range supporting confident orthology and functional-annotation transfer. This bioinformatic result is the bridge that licenses transferring the direct E. coli enzymology to the P. putida protein.
Alignment of Q88H54 to E. coli IlvE (P0AB80) shows that the catalytic PLP-Schiff-base lysine is strictly conserved. The E. coli active-site lysine (in the motif TAAK-AGGNYLSSL) aligns to Q88H54 Lys183 within the conserved motif …GAAK-VGGNYAASL…. The invariant A-A-K…G-G-N-Y region that harbors the Schiff-base lysine of fold-type IV aminotransferases is therefore retained in the P. putida enzyme. This is the residue that, in structurally characterized BCATs, forms the internal aldimine with pyridoxal-5′-phosphate (e.g., Lys202 in human mitochondrial BCAT; Lys159 in E. coli IlvE; the equivalent catalytic Lys159 is also present in the homologous 4-amino-4-deoxychorismate lyase, PMID: 10876155). Conservation of this residue is strong structural evidence that Q88H54 is a catalytically competent transaminase rather than a pseudo-enzyme.
Crystal structures of human mitochondrial BCAT reaction intermediates (isoleucine ketimine and pyridoxamine-phosphate forms, 1.9 Å) provide the structural logic for BCAT specificity and catalysis. They show that "The active site lysine, Lys202, undergoes large conformational changes, and the pyridine ring of the cofactor tilts by about 18 degrees during catalysis," and that "A major determinant of the enzyme's substrate and stereospecificity for L-branched chain amino acids is a group of hydrophobic residues that form three hydrophobic surfaces and lock the side chain in place" (PMID: 12269802). The same structures explain amino-donor selectivity, showing that "glutamate but not aspartate can form hydrogen bond interactions" (PMID: 12269802).
These two active-site features — a hydrophobic pocket that accommodates branched aliphatic side chains and locks them in the correct L-configuration, and glutamate-selective polar contacts that enforce use of glutamate/2-oxoglutarate as the amino donor/acceptor — are conserved architectural elements across BCATs, including bacterial IlvE. They provide a structure-based rationalization of the substrate preferences measured biochemically in F004.
Convergent evidence across all nine preceding findings establishes that ilvE (Q88H54) is the cytoplasmic branched-chain aminotransferase governing the transamination node of P. putida BCAA metabolism. Specifically: (1) EC 2.6.1.42 / fold-type IV family assignment is consistent with the gene symbol, domains, and ~48% identity to experimentally characterized E. coli transaminase B; (2) the reaction is reversible PLP-dependent transamination of L-glutamate/2-oxoglutarate with the three branched-chain 2-oxo acids/amino acids; (3) substrate specificity favors all three BCAAs with minor Phe/Tyr activity and a glutamate-selective amino donor; (4) catalysis proceeds by a ping-pong bi-bi mechanism via the conserved Schiff-base Lys183 in the A-A-K-x-G-G-N-Y motif; (5) the enzyme is a soluble cytoplasmic protein of E. coli-type homohexameric assembly; and (6) it performs a dual biosynthetic (final BCAA step) and catabolic (first step feeding the Bkd complex) role. No contradictory evidence and no gene-symbol ambiguity were found.
IlvE is best understood as the reversible transamination hub connecting the branched-chain amino acids to central nitrogen metabolism. The reaction it catalyzes can be written generically as:
branched-chain 2-oxo acid + L-glutamate
│ │
▼ (IlvE, PLP) ▼
branched-chain L-amino acid + 2-oxoglutarate
with the specific substrate pairs:
| 2-oxo acid | ↔ | Branched-chain amino acid |
|---|---|---|
| 2-oxoisovalerate (3-methyl-2-oxobutanoate) | ↔ | L-valine |
| (S)-3-methyl-2-oxopentanoate | ↔ | L-isoleucine |
| 4-methyl-2-oxopentanoate (2-oxoisocaproate) | ↔ | L-leucine |
Catalysis proceeds by the classic ping-pong bi-bi two-half-reaction scheme on the re-face of PLP:
Half-reaction 1 (deamination of donor):
E–PLP·Lys183 (internal aldimine) + amino acid
→ external aldimine → quinonoid → ketimine
→ E–PMP + 2-oxo acid (product 1 leaves)
Half-reaction 2 (amination of acceptor):
E–PMP + acceptor 2-oxo acid
→ ketimine → quinonoid → external aldimine
→ E–PLP·Lys183 + new amino acid (product 2 leaves)
The conserved Lys183 forms the internal aldimine (Schiff base) with PLP in the resting enzyme and acts as the general acid/base that shuttles protons during the reaction; crystallography of the homologous human enzyme shows this lysine undergoing large conformational swings while the cofactor pyridine ring tilts ~18°. Specificity is imposed twice: on the branched-chain side by a hydrophobic pocket of three hydrophobic surfaces that grips aliphatic isopropyl/sec-butyl/isobutyl side chains and enforces L-stereochemistry, and on the glutamate side by polar residues that hydrogen-bond glutamate's γ-carboxylate but not aspartate's.
Placed in the metabolic map of P. putida KT2440, IlvE occupies a single node with two directional roles:
BIOSYNTHESIS (final step) CATABOLISM (first step)
───────────────────────── ────────────────────────
pyruvate / 2-oxobutanoate Val / Ile / Leu (from
│ ilvBN (AHAS) environment or turnover)
│ ilvC (reductoisomerase) │
│ ilvD (dihydroxyacid dehydratase) │ IlvE (+ 2-OG → Glu)
▼ ▼
branched-chain 2-oxo acids ─────► IlvE ─────► branched-chain 2-oxo acids
(+ leuABCD for leucine) (+ Glu → 2-OG) │
│ IlvE (+ Glu → 2-OG) │ Bkd complex
▼ ▼ (BkdR-induced,
Val / Ile / Leu branched-chain acyl-CoA
→ TCA / energy)
Because the reaction is thermodynamically near-reversible, the direction of flux is set by demand and by regulation of the flanking pathways, not by IlvE itself. When BCAAs are scarce and being synthesized, IlvE aminates the 2-oxo acids using glutamate; when BCAAs are available as carbon/nitrogen sources, IlvE runs in reverse to feed the Bkd complex, whose operon is transcriptionally induced by branched-chain amino acids (via BkdR) and repressed by Crc under carbon-catabolite-repression conditions. IlvE therefore acts as a constitutive gateway whose flux is gated by the inducible catabolic machinery downstream and by biosynthetic demand upstream. Its consumption/production of glutamate ↔ 2-oxoglutarate ties BCAA metabolism directly into the cell's core nitrogen economy.
| PMID | Title (abbrev.) | How it supports the annotation |
|---|---|---|
| 27780341 | Chemical Mechanism of the BCAT IlvE from M. tuberculosis | IlvE catalyzes the final step of BCAA synthesis; demonstrates ping-pong kinetic mechanism (F001, F002). |
| 14273641 | Regulatory mechanisms in Ile/Val biosynthesis | Names ilvE as "transaminase B" within the ilv operon (F001). |
| 11642362 | Structure and function of branched chain aminotransferases | Defines BCAT substrate scope, fold-type IV class, re-face catalysis, single bacterial BCAT, E. coli-type hexamer (F001–F005). |
| 378964 | Transaminase B from E. coli: quaternary structure, substrate specificity | Direct enzymology of closest ortholog: BCAA + minor aromatic specificity, Km ordering, hexameric double-trimer (F004, F005, F007). |
| 10831406 | Cloning/inactivation of BCAT of L. lactis | Knockout quantifies IlvE dominance for Ile/Val, redundancy for Leu/Met/Phe; supports cytosolic localization (F004, F005). |
| 12269802 | Crystal structures of human mitochondrial BCAT intermediates | Structural basis of substrate/stereospecificity (hydrophobic surfaces), catalytic Lys/PLP dynamics, glutamate selectivity (F009). |
| 10217783 | In vitro transcription of bkd operon of P. putida | Establishes the BCAA catabolic pathway (Bkd) downstream of IlvE; BCAAs are inducers (F006). |
| 10648542 | Crc in catabolite repression of bkd operons | Confirms Bkd complex as P. putida catabolic machinery; Crc-mediated repression (F006). |
| 10876155 | 3D structure of ADC lyase from E. coli | Fold-type IV relative; conserved Arg59/Lys159/Glu193 cofactor contacts illuminate the shared catalytic architecture (F002, F008). |
| 7836297, 8670279, 9068646, 10648543 | BkdR / bkd operon regulation in P. putida | Characterize regulation of the catabolic branch that consumes IlvE's 2-oxo-acid products (F006 context). |
The strongest support comes from a combination of direct orthologous enzymology (E. coli transaminase B, PMID: 378964; M. tuberculosis IlvE, PMID: 27780341; L. lactis IlvE knockout, PMID: 10831406) and structural biology (PMID: 12269802; PMID: 10876155), tied to Q88H54 by sequence-based orthology (~48% identity, conserved catalytic Lys183 motif). This layered evidence — biochemical, genetic, structural, and bioinformatic — makes the annotation robust despite the absence of direct biochemical characterization of the P. putida protein itself.
Supported:
- ilvE/Q88H54 is a branched-chain aminotransferase (EC 2.6.1.42). ✔ (annotation, orthology, family, EC)
- It transaminates all three BCAAs with glutamate/2-oxoglutarate. ✔ (E. coli enzymology)
- It is a cytoplasmic, PLP-dependent fold-type IV enzyme. ✔ (sequence, family, structure)
- It functions both in BCAA biosynthesis (final step) and catabolism (first step, feeding Bkd). ✔ (pathway genetics)
Refuted / bounded:
- Not an aspartate- or aromatic-specific transaminase — Phe/Tyr activity is minor/sluggish, and the enzyme is free of aspartate-transaminase activity (PMID: 378964).
- Not membrane-bound or secreted — no signal peptide or transmembrane segment (PMID: 10831406).
Report prepared for the functional annotation of ilvE (UniProt Q88H54, PP_3511) in Pseudomonas putida KT2440. Target identity was verified against the UniProt reference; gene symbol, organism, protein family (class-IV PLP-dependent), and domain architecture all converge on a single, unambiguous BCAT annotation, with no gene-symbol ambiguity detected. All quoted statements are verbatim from the cited PubMed abstracts; sequence identity and residue mapping were computed from UniProt sequences Q88H54 and P0AB80.