Information on EC 4.3.1.7 - ethanolamine ammonia-lyase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
4.3.1.7
-
RECOMMENDED NAME
GeneOntology No.
ethanolamine ammonia-lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ethanolamine = acetaldehyde + NH3
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Deamination
elimination
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ethanolamine utilization
-
-
Glycerophospholipid metabolism
-
-
Metabolic pathways
-
-
SYSTEMATIC NAME
IUBMB Comments
ethanolamine ammonia-lyase (acetaldehyde-forming)
A cobalamin protein.
CAS REGISTRY NUMBER
COMMENTARY hide
9054-69-7
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
Q720Q3;
involved in ethanolamine degradation pathway
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2R)-2-aminopropanol
?
show the reaction diagram
-
deamination with inversion of configuration
-
-
?
(2S)-2-aminopropanol
propionaldehyde + ?
show the reaction diagram
-
deamination with retention of configuration
-
?
(R)-2-aminopropanol
propanal + NH3
show the reaction diagram
-
-
-
-
?
(S)-1-amino-2-propanol
propanal + NH3
show the reaction diagram
-
inactive substrate analogue, which binds to the substrate binding site in EAL but does not form the cob(II)alamin-substrate radical pair state
-
-
?
(S)-2-aminopropanol
propanal + NH3
show the reaction diagram
ethanolamine
?
show the reaction diagram
-
first enzyme in ethanolamine degradation
-
-
-
ethanolamine
acetaldehyde + NH3
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ethanolamine
?
show the reaction diagram
-
first enzyme in ethanolamine degradation
-
-
-
ethanolamine
acetaldehyde + NH3
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
adenosylcobalamin
cobamide
coenzyme B12
vitamin B12
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NH4+
-
can replace K+
Rb+
-
can replace K+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(S)-1-amino-2-propanol
-
inactive substrate analogue
1-amino-2-propanol
-
-
2-(dimethylamino)ethanol
-
-
2-(methylamino)ethanol
-
-
2-amino-1-butanol
-
-
2-amino-2-methyl-1-propanol
-
-
3-aminopropanol
-
-
adenosylmethylcobalamin
-
0.1% coenzyme activity compared to adenosylcobalamin, holoenzyme with adenosylmethylcobalamin undergoes rapid inactivation
cobamide coenzyme
-
inactivated in absence of substrate
cyanocobalamin
DL-1,3-diaminopropan-2-ol
-
competitive
DL-1-aminopropan-2-ol
-
competitive
DL-2-amino-1-propanol
-
-
ethanolamine
-
10 mM
ethylene glycol
-
inactivates the EAL holoenzyme
hydroxycobalamin
hydroxyethylhydrazine
-
the latter suicide inhibitor effects a stoichiometric conversion of enzyme-bound adenosylcobalamin into its cleaved form cob(II)alamin
iodoacetamide
L-2-Aminopropan-1-ol
methanol
-
inactivates the EAL holoenzyme
methylcobalamin
p-chlormercuribenzoate
-
-
p-Chloromercuriphenylsulfonate
-
-
p-hydroxymercuribenzoate
-
0.0001 mM, 50% inhibition; even in the presence of ethanolamine
Urea
-
above 1 mM
additional information
-
the holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. Inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+ and free adensosylcobalamin. EutA is essential for reactivation. Reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
tris-(2-carboxyethyl) phosphine
leads to 25fold increase in kcat
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0008 - 0.17
(S)-2-aminopropanol
0.000055
adenosylcobalamin
-
wild-type enzyme
0.0019 - 2
ethanolamine
additional information
adenosylcobalamin
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.036 - 770
ethanolamine
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
720 - 730
ethanolamine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.039
(S)-1-amino-2-propanol
-
dissociation constant for interaction with holo-EAL
0.0001
p-hydroxymercuribenzoate
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.07
iodoacetamide
Escherichia coli;
-
wild-type enzyme
0.0003
p-chlormercuribenzoate
Escherichia coli;
-
wild-type enzyme
additional information
iodoacetamide
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55.1
-
wild-type enzyme, after purification
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8 - 8.9
-
pH 5.8: about 30% of maximal activity, pH 8.9: about 70% of maximal activity
6.6 - 8.2
-
broad pH-optimum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
cytosolic organelles, bacterial microcompartment
Manually annotated by BRENDA team
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carboxysome-like structure, is nedded to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA
-
Manually annotated by BRENDA team
additional information
-
microcompartiment shell protein
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
UNIPROT
ORGANISM
Escherichia coli (strain K12);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32100
-
eutC protein subunit contains 286 amino acids
35000
-
alpha6beta6, 6 * 35000 + 6 * 50000, SDS-PAGE
35200
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6 * 35200 + 6 * 56900, the small subunit seems to be responsible for cobalamin binding, SDS-PAGE
36000
-
x * 36000 + x * 51000, the 2 subunits are probably present in equimolar proportions, SDS-PAGE
49400
-
eutB protein subunit contains 453 amino acids and the active site
51000
-
x * 36000 + x * 51000, the 2 subunits are probably present in equimolar proportions, SDS-PAGE
56900
-
6 * 35200 + 6 * 56900, the small subunit seems to be responsible for cobalamin binding, SDS-PAGE
57000
-
8-10 * 57000, equilibrium sedimentation after treatment with guanidinium hydrochloride
66800
-
for the trimer, determination by gel filtration
500000
520000
560400
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
heterodecamer
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alphabeta, 6*49000, 6*32000, SDS-PAGE
hexamer
-
functional protein is a hexamer of alpha-beta-dimers (alpha-subunit of 50000 Da and beta-subunit of 31000 Da)
oligomer
trimer
-
determination by gel filtration, freshly prepared protein oligomerizes readily into trimers in the presence or absence of 5 mM beta-mercaptoethanol, monomer consists of 219-amino-acids
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
side-chain modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
deletion mutants of the enzyme beta subunit DELTA4-30 and DELTA4-43, to 8.0 and 2.1 A resolution, respcetively
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For crystallization trials, protein sample is dialyzed against 50 mM HEPES with pH 7.0 and concentrates to a final concentration of about 1 mg/ml. Purification results in highly pure protein that crystallizes readily under many different conditions, protein forms thin hexagonal plate-shaped crystals belonging to space group P3. Best crystals of Eut-L_NHIS are obtained in 3.3 M ammonium acetate, 5% polyethylene glycol 400 and 50 mM Tris buffer pH 7.5, crystals grow as hexagonal plates. Eut-L_CHIS crystals grow as single hexagonal plates with sharp edges. Crystals grow in 2 M NaCl, 100 mM phosphate, 100 mM MES buffer pH 6.5 and 4% PEG 400.
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N-terminal His6-tagged beta subunit lacking residues Lysbeta4-Cysbeta43, complexed with CN-cobalamin and (R)-2-amino-1-propanol or (S)-2-amino-1-propanol. The lower affinity for the (R)-enantiomer may be due to the conformational change of the ValR326 side chain of the enzyme. The pro-S hydrogen atom on C1 is abstracted by the adenosyl radical from both enantiomeric substrates. The NH2 group migrates from C2 to C1 by a suprafacial shift, with inversion of configuration at C1 for both enantiomeric substrates. (R)-2-amino-1-propanol is deaminated by the enzyme with inversion of configuration at C2, whereas the (S)-enantiomer is deaminated with retention. The rotameric radical intermediate from the (S)-enantiomer undergoes flipping to the rotamer from the (R)-enantiomer before the hydrogen back-abstraction, suggesting the preference of the enzyme active site for the rotamer from the (R)-enantiomer in equilibration, partly explained by steric repulsion of the (S)-enantiomer-derived product radical at C3 with the PheR329 and LeuR402 residues
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N-terminal His6-tagged beta subunit lacking residues Lysbeta4-Cysbeta43, in complex with cyanocobalamin and in complex with cyanocobalamin or adeninylpentylcobalamin and substrates. The enzyme exists as a trimer of the (alphabeta)2 dimer. The active site is in the (beta/alpha)8 barrel of the-subunit, the beta-subunit covers the lower part of the cobalamin that is bound in the interface of the alpha- and beta-subunits. The structure complexed with adeninylpentylcobalamin reveals the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Arg160 contributes to substrate binding most likely by hydrogen bonding with the O1 atom. Marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme-Co-C bond. A major structural change upon substrate binding is not observed with this particular enzyme. Glu287, one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co-C bond
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 8.5
-
crystals have a remarkable high stability with respect to changes in pH
690280
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ethanolamine, dithiothreitol, glycerol and KCl protect the apoenzyme from inactivation
-
K+ stabilizes the enzyme during dialysis or storage
-
unusually high stability against different solvent conditions with respect to changes in pH and ionic strength.
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, Tris/HCl buffer supplemented with glycerol, dithiothreitol, KCl and ethanolamine, stable for several months
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4°C, 24 h, stable
-
4°C, enzyme concentration 4 mg/ml, 10% loss of activity after 8 days, considerably less stable at concentrations below 0.1 mg/ml
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
31% yield after DEAE-cellulose column
-
by nickel-affinity chromatography
-
N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties
-
using Ni-NTA chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and overexpression of three different versions (eut-LpET151 his-tagged version (appends a cleavable 33-amino acid tag sequence to the N-terminus), Eut-L_NHIS (short His6-tagged version) and Eut-L_CHIS (short His6-tagged version)) of the protein is carried out directly from the Escherichia coli genome by selective PCR amplification. Selenomethione-derivatized proteins are obtained by growing cloned bacteria in selenomethionine-containing M9 minimal media. Protein overexpression and purification are performed.
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cloning in Escherichia coli overexpression strain incorporating the cloned Salmonella typhimurium EAL coding sequence
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Escherichia coli overexpression strain incorporating the cloned Salmonella typhimurium EAL coding sequence
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expressed in Escherichia coli as a His-tagged fusion protein
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expression in Escherichia coli
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expression in Escherichia coli overexpression strain incorporating the cloned Salmonella typhimurium EAL coding sequences
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N-terminal His6-tagged beta subunit lacking residues Lysbeta4-Cysbeta43
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The plasmid containing the ethanolamine ammonia-lyase coding sequence is extracted from the Escherichia coli overexpression strain. Site-directed mutagenesis is performed on the R160 position within the eutb sequence by using the GeneTailor site-directed mutagenesis kit. Five plasmids, including the four mutations R160A, R160K, R160E, and R160I and a wild-type control, are constructed and transformed into the competent Escherichia coli DH5R T1R strain.
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the plasmid pET-SEAL, encoding the small and large subunits of EAL is expressed in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D362A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
D362E
-
mutant shows highly decreased kcat compared to wild-type
D362N
-
mutant shows highly decreased kcat compared to wild-type
E287A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
E287D
-
mutant shows highly decreased kcat compared to wild-type
E287H
-
mutant shows highly decreased kcat compared to wild-type
E287Q
-
mutant shows highly decreased kcat compared to wild-type
N193A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
N193D
-
mutant shows decreased kcat compared to wild-type but mutant retains partial activity, Km (ethanolamine) increased compared to wild-type
Q162A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
Q162E
-
mutant shows decreased kcat compared to wild-type but mutant retains partial activity, Km (ethanolamine) increased compared to wild-type
Q162H
-
mutant shows decreased kcat compared to wild-type but mutant retains partial activity, Km (ethanolamine) increased compared to wild-type
Q162K
-
mutant shows highly decreased kcat compared to wild-type
R160A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
R160K
-
mutant shows decreased kcat compared to wild-type but mutant retains partial activity, Km (ethanolamine) increased compared to wild-type
R160A
-
Assembles into an oligomer, catalytically inactive, reacts with substrates to form magnetically isolated Co2+ and unidentified radical species, activity is resurrected by externally added guanidinium to 2.3% of wild-type. R160A EutB is unstable and forms strongly associated, high molecular mass aggregates.
R160E
-
mutant fails to assemble into an oligomer
R160I
-
mutant fails to assemble into an oligomer
R160K
-
assembles into an oligomer, mutant displays catalytic turnover of aminoethanol, with a 180-fold lower value of kcat/KM relative to wild-type enzyme, forms Co2-substrate radical pair intermediate states during turnover on aminoethanol and (S)-2-aminopropanol substrates
additional information
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