| Field | Value |
|---|---|
| UniProt | Q88QF2 |
| Gene | eutC (ordered locus PP_0542) |
| Organism | Pseudomonas putida KT2440 (ATCC 47054 / DSM 6125 / NCIMB 11950) |
| Protein | Ethanolamine ammonia-lyase (EAL) small subunit; EC 4.3.1.7 |
| Length | 272 aa |
| Domains | Pfam PF05985 (EutC); InterPro EutC (IPR009246), EutC_N (IPR042255), EutC_C (IPR042251) |
| Partner gene | eutB (PP_0543), EAL large subunit (Q88QF1) |
Verification passed. The gene symbol eutC, the EutC/PF05985 domain set, the EC number 4.3.1.7, and the immediately adjacent eutB large-subunit gene all agree with the UniProt-provided identity. The literature on "EutC" as the ethanolamine ammonia-lyase small subunit is directly on-target; no gene-symbol ambiguity was encountered. Direct biochemical/structural characterization exists for the E. coli and Salmonella orthologues; the P. putida protein is annotated by homology (HAMAP rule MF_00601), and I add a P. putida-specific genomic-context analysis below.
EutC is the small (β) subunit of ethanolamine ammonia-lyase (EAL, EC 4.3.1.7), an adenosylcobalamin (coenzyme B12)-dependent enzyme. Working as an obligate heterodimer with the large subunit EutB, EAL catalyzes the deamination of ethanolamine to acetaldehyde + ammonia by radical chemistry. EutC itself does not contain the catalytic TIM barrel; rather it partners with EutB to complete the cobalamin-binding pocket at the subunit interface and is required for a functional enzyme. In P. putida KT2440 the enzyme lets the cell use ethanolamine as a carbon and nitrogen source (given exogenous/endogenous B12), operating as a soluble cytoplasmic enzyme within a minimal, microcompartment-independent ethanolamine-utilization gene cluster.
Reaction (EC 4.3.1.7, Rhea:15313):
ethanolamine → acetaldehyde + NH4⁺
EAL is the committed, first catabolic step of ethanolamine degradation. Comparative genomics states plainly that "ethanolamine-ammonia lyase, the enzyme that breaks ethanolamine into acetaldehyde and ammonia, is encoded by the gene tandem eutBC" (Tsoy et al., 2009, PMID 19783625). The products feed central metabolism: the ammonia serves as a nitrogen source, and acetaldehyde is oxidized (by the neighboring aldehyde dehydrogenase, below) toward acetyl-CoA/acetate as a carbon and energy source.
Substrate specificity. The physiological substrate is ethanolamine (2-aminoethanol). The family is characterized as deaminating "various vicinal amino-alcohols to oxo compounds" (UniProt FUNCTION, MF_00601), i.e. it is a vicinal-amino-alcohol ammonia-lyase; ethanolamine is the canonical and best substrate. The enzyme also deaminates both enantiomers of 2-amino-1-propanol (2-aminopropanol), non-stereospecifically; enantiomer-bound crystal structures rationalize the lower affinity for the (R)-form via a Val-α326 side-chain conformational change and predict pro-S hydrogen abstraction at C1 (Shibata et al., 2011, PMID 21142024), consistent with ¹⁵N-isotope-effect/EPR studies on ethanolamine and (R/S)-2-aminopropanol (Poyner et al., 2006, PMID 16734439). The specificity is thus narrow — small 1,2-amino alcohols. Crystallographic work shows the substrate is held by six hydrogen bonds to active-site residues, with Arg(α160) contributing binding through the substrate O1 hydroxyl (Shibata et al., 2010, PMID 20519496) — the spectator hydroxyl group is mechanistically essential (Wetmore et al., 2002, PMID 12440904).
Note on division of labor. The catalytic and substrate/cobalamin-binding residues lie on the large subunit EutB. Site-directed mutagenesis shows Arg160 of EutB is critical for both oligomer assembly and radical catalysis (R160A is inactive; R160K lowers k꜀ₐₜ/K_M ~180-fold) (Sun et al., 2008, PMID 18444665). This reinforces that EutC is the non-catalytic structural partner that completes the interfacial cobalamin site.
EAL is a class II (adenosylcobalamin-dependent) isomerase/eliminase. Its cofactor is adenosylcob(III)alamin (coenzyme B12; ChEBI:18408), which UniProt notes "binds between the large and small subunits."
The chemistry is radical-based, not hydrolytic:
1. Homolytic cleavage of the cobalt–carbon bond of AdoCbl generates a 5′-deoxyadenosyl radical (Frey, 2001, PMID 11395404).
2. This radical abstracts a hydrogen atom from ethanolamine, producing a substrate radical.
3. The substrate radical undergoes a 1,2-amino-group shift to a carbinolamine radical, which collapses to acetaldehyde + ammonia (Wetmore et al., 2002, PMID 12440904).
Experimental EPR of purified EAL confirms "the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode," i.e. with 5,6-dimethylbenzimidazole coordinating the cobalt (Akita et al., 2010, PMID 19762342).
Quaternary structure. The functional unit is a EutB·EutC heterodimer (α·β) that assembles into a heterododecamer (EutB)6(EutC)6 — "the enzyme existed as the heterododecamer α6β6" (Akita et al., 2010, PMID 19762342). Structurally it is a "trimer of the (αβ)2 dimer," with "6 large subunits form[ing] a core ring with 6 small subunits projecting outwards" (UniProt SUBUNIT; Shibata et al., 2010).
What EutC does structurally. The catalytic machinery — the (β/α)8 TIM barrel that holds the active site — resides in the large subunit EutB. The small subunit EutC "covers the lower part of the cobalamin that is bound in the interface of the α- and β-subunits" (Shibata et al., 2010, PMID 20519496). Thus EutC:
- forms the obligate protein partner of EutB (both genes are required for activity),
- helps build and cap the cobalamin-binding site at the subunit interface,
- is present in 1:1 stoichiometry with EutB, consistent with the finding that binding one cobalamin-analog inhibitor per αβ unit completely inhibits the enzyme (Akita et al., 2010).
Domain architecture / evolution. EutC arose by recruitment of a Rossmann-fold domain fused to an N-terminal α-helical domain (EutB is the paired TIM-barrel + N-terminal helical subunit) (Tsoy et al., 2009, PMID 19783625) — matching the InterPro EutC_N/EutC_C two-domain layout of Q88QF2. The N-terminal region is dispensable for catalysis: N-terminally truncated EAL retains catalytic properties, and "a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme" (Akita et al., 2010).
EAL is a cytoplasmic (soluble) enzyme. In enterobacteria such as Salmonella and E. coli, EAL is encapsulated inside a bacterial microcompartment (metabolosome) — a proteinaceous organelle whose shell (e.g., EutL, PMID 19451619) confines the volatile/toxic intermediate acetaldehyde (Sturms et al., 2015, PMID 25962913). UniProt's HAMAP-inferred "Bacterial microcompartment" localization for Q88QF2 is transferred from these organisms.
However, a P. putida KT2440-specific caveat applies. Genomic-neighborhood analysis (this work) shows the KT2440 eut cluster is minimal — eutC (PP_0542), eutB (PP_0543), an ethanolamine transporter (PP_0544, EutH-like), an aldehyde dehydrogenase (PP_0545, aldB-I, EutE-like), and a σ54-dependent regulator (PP_0546) — and contains no microcompartment shell-protein genes. A genome-wide UniProt proteome search (this work) confirms this: Pfam PF00936 = 0 hits, Pfam PF03319 = 0 hits, keyword "carboxysome" = 0, and keyword "microcompartment" returns only eutC/eutB themselves. Therefore P. putida KT2440 builds no bacterial microcompartment, and EutBC functions as a free cytoplasmic enzyme rather than inside a BMC. The HAMAP-inferred microcompartment localization is an over-transfer from enterobacteria and should be regarded as inapplicable to this organism.
Cofactor supply is intrinsic. Although UniProt phrases activity as requiring "external vitamin B12," KT2440 encodes cobalamin biosynthesis/salvage genes (cobN PP_3507, cobS PP_1681, cobD PP_1675, cobE PP_3409) and — critically — the corrinoid adenosyltransferase PduO (PP_1349, EC 2.5.1.17) that generates adenosylcobalamin, the exact AdoCbl coenzyme EutBC requires, plus periplasmic cobalamin transport proteins (PP_2418; HutB PP_0524). The organism can therefore both import and adenosylate B12 to feed EAL.
EutC functions in amine degradation / ethanolamine catabolism (UniProt PATHWAY). The reconstructed minimal pathway from the gene cluster:
Ethanolamine itself is a widespread nutrient derived from turnover of the membrane phospholipid phosphatidylethanolamine (Kaval & Garsin, 2018, PMID 29463652); catabolizing it lets P. putida scavenge additional carbon and nitrogen from its (soil/rhizosphere and host-associated) environment.
This "stripped-down," microcompartment-free architecture is consistent with the variable eut gene content across bacteria, including loss/absence of auxiliary genes in many lineages (Tsoy et al., 2009, PMID 19783625).
Supported
- EutC is the small subunit of AdoCbl-dependent EAL (EC 4.3.1.7) catalyzing ethanolamine → acetaldehyde + NH3. (UniProt; PMID 19783625, 19762342)
- EutC is an obligate structural partner of EutB, capping the interfacial cobalamin site in an (EutB)6(EutC)6 dodecamer. (PMID 20519496, 19762342)
- Catalysis proceeds via AdoCbl Co–C homolysis → 5′-deoxyadenosyl radical → 1,2-amino shift. (PMID 11395404, 12440904, 19762342)
- EutC = Rossmann-fold + N-terminal α-helical domain; N-terminus dispensable for catalysis. (PMID 19783625, 19762342)
- Substrate range is narrow (small 1,2-amino alcohols): ethanolamine (physiological) plus both enantiomers of 2-aminopropanol; catalytic residues (Arg160) reside on EutB, confirming EutC's structural (non-catalytic) role. (PMID 21142024, 16734439, 18444665)
Refuted / corrected for this organism
- The HAMAP-inferred "bacterial microcompartment" localization is not supported for P. putida KT2440. A genome-wide proteome scan finds no BMC shell-protein genes (Pfam PF00936 = 0, PF03319 = 0; "carboxysome" = 0), so EutC acts in the cytoplasm. (genome-wide bioinformatic analysis; contrast with PMID 19451619, 25962913)
- The "requires external vitamin B12" wording is only partially apt: KT2440 encodes cobalamin biosynthesis/salvage plus the PduO adenosyltransferase (PP_1349) that makes the AdoCbl coenzyme, so cofactor provision is intrinsic. (bioinformatic analysis)