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
-
-
SYSTEMATIC NAME
IUBMB Comments
ethanolamine ammonia-lyase (acetaldehyde-forming)
A cobalamin protein.
CAS REGISTRY NUMBER
COMMENTARY hide
9054-69-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
enzyme activity rapidly declines in the stationary phase of growth
-
-
Manually annotated by BRENDA team
-
Q720Q3
Uniprot
Manually annotated by BRENDA team
strain N186/21
-
-
Manually annotated by BRENDA team
strain N186/21
-
-
Manually annotated by BRENDA team
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
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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
P19264 and P19265
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
0.067
(R)-2-aminopropanol
Salmonella enterica subsp. enterica serovar Typhimurium
-
-
0.12
(S)-2-aminopropanol
Salmonella enterica subsp. enterica serovar Typhimurium
-
-
0.036 - 770
ethanolamine
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
720 - 730
ethanolamine
456
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
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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
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Manually annotated by BRENDA team
additional information
-
microcompartiment shell protein
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
P19264 and P19265
SDS-PAGE, EutC monomer
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
48000
P19264 and P19265
SDS-PAGE, EutB monomer
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
494000
P19264 and P19265
native PAGE, EAL oligomer
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
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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
-20C, Tris/HCl buffer supplemented with glycerol, dithiothreitol, KCl and ethanolamine, stable for several months
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4C, 24 h, stable
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4C, enzyme concentration 4 mg/ml, 10% loss of activity after 8 days, considerably less stable at concentrations below 0.1 mg/ml
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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 as a His-taggd fusion protein
P19264 and P19265
expressed in Escherichia coli as a His-tagged fusion protein
-
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
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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
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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
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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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