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Information on EC 4.3.1.7 - ethanolamine ammonia-lyase and Organism(s) Escherichia coli and UniProt Accession P19636
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4.3.1.7 ethanolamine ammonia-lyaseIUBMB Comments
A cobalamin protein.
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Word Map
- 4.3.1.7
-
b12-dependent
- adenosylcobalamin
-
adenosylcobalamin-dependent
-
cobiialamin
- 5'-deoxyadenosine
-
homolysis
- cobamide
-
corrin
-
5\'-deoxyadenosyl
-
adocbl
-
adocbl-dependent
-
cobalt-carbon
- aminoethanol
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x-band
-
full-spectrum
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metabolosome
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2h-labeled
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carbon-cobalt
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cryotrapped
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cryosolvent
- 2-aminoethanols
- corrinoids
-
eseem
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
ethanolamine ammonia-lyase, ethanolamine ammonia lyase, eutbc, ethanolamine deaminase, ethanolamine-ammonia lyase, eut-l, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
ammonia-lyase, ethanolamine
-
-
-
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EAL
ethanolamine deaminase
-
-
-
-
EAL
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
ethanolamine ammonia-lyase (acetaldehyde-forming)
A cobalamin protein.
CAS REGISTRY NUMBER
COMMENTARY
9054-69-7
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes
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-
?
ethanolamine
acetaldehyde + NH3
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Escherichia coli has evolved specialized organelles (microcompartments) for the degradation of small molecular compounds such as ethanolamine and propanediol
-
-
?
ethanolamine
acetaldehyde + NH3
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assay at pH 8.0, 37°C, reaction terminated by adding potassium citrate buffer
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ethanolamine
acetaldehyde + NH3
-
Escherichia coli has evolved specialized organelles (microcompartments) for the degradation of small molecular compounds such as ethanolamine and propanediol
-
-
?
additional information
?
-
-
ethanolamine ammonia-lyase microcompartment is composed of five different shell proteins that have been proposed to assemble into symmetrically shaped polyhedral particles of varying sizes
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
adenosylcobalamin
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
adenosylcobalamin
-
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
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
adenosylmethylcobalamin
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0.1% coenzyme activity compared to adenosylcobalamin, holoenzyme with adenosylmethylcobalamin undergoes rapid inactivation
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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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
adenosylcobalamin
-
truncated enzymes, value between 0.000041 and 0.000078
additional information
ethanolamine
-
truncated enzymes, value between 0.0059 and 0.0076
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
iodoacetamide
Escherichia coli
-
truncated enzymes, value between 0.07 to 0.24
additional information
p-chlormercuribenzoate
Escherichia coli
-
truncated enzymes, value around 0.0013
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
truncated enzymes, after purificaton, value between 12.45 and 63.1
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
35200
-
6 * 35200 + 6 * 56900, the small subunit seems to be responsible for cobalamin binding, SDS-PAGE
56900
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6 * 35200 + 6 * 56900, the small subunit seems to be responsible for cobalamin binding, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dodecamer
-
6 * 35200 + 6 * 56900, the small subunit seems to be responsible for cobalamin binding, SDS-PAGE
trimer
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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
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
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
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
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D362A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
E287A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
N193A
-
mutant shows highly decreased kcat compared to wild-type, mutant undergoes quicker inactivation compared to wild-type
N193D
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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
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
additional information
additional information
Overexpression of deletion mutants of the enzyme beta subunit DELTA4-30 and DELTA4-43. N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties
additional information
-
Overexpression of deletion mutants of the enzyme beta subunit DELTA4-30 and DELTA4-43. N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties
additional information
-
N-terminal truncated enzyme, similar to wild-type enzyme in catalytic properties, more resistant to p-chloromercuribenzoate than wild-type enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
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
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unusually high stability against different solvent conditions with respect to changes in pH and ionic strength.
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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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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
N-terminal His6-tagged beta subunit lacking residues Lysbeta4-Cysbeta43
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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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Scarlett, F.A.; Turner, J.M.
Microbial metabolism of amino alcohols. Ethanolamine catabolism mediated by coenzyme B12-dependent ethanolamine ammonia-lyase in Escherichia coli and Klebsiella aerogenes
J. Gen. Microbiol.
95
173-176
1976
Escherichia coli, Klebsiella aerogenes
Pennington, S.J.; Jones, P.W.; Turner, J.M.
The adenosylcobalamin-dependent ethanolamine ammonia-lyase of Escherichia coli
Biochem. Soc. Trans.
9
447
1981
Escherichia coli
-
Blackwell, C.M.; Scarlett, F.A.; Turner, J.M.
Ethanolamine catabolism by bacteria, including Escherichia coli
Biochem. Soc. Trans.
4
495-497
1976
Klebsiella aerogenes, Leifsonia aquatica, Escherichia coli
Blackwell, C.M.; Turner, J.M.
Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lyase of Escherichia coli
Biochem. J.
175
555-563
1978
Escherichia coli
Fukuoka, M.; Nakanishi, Y.; Hannak, R.B.; Krautler, B.; Toraya, T.
Homoadenosylcobalamins as probes for exploring the active sites of coenzyme B12-dependent diol dehydratase and ethanolamine ammonia-lyase
FEBS J.
272
4787-4796
2005
Escherichia coli
Mori, K.; Bando, R.; Hieda, N.; Toraya, T.
Identification of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase
J. Bacteriol.
186
6845-6854
2004
Escherichia coli
Nikolakakis, K.; Ohtaki, A.; Newton, K.; Chworos, A.; Sagermann, M.
Preliminary structural investigations of the Eut-L shell protein of the ethanolamine ammonia-lyase metabolosome of Escherichia coli
Acta Crystallogr. Sect. F
65
128-132
2009
Escherichia coli
Akita, K.; Hieda, N.; Baba, N.; Kawaguchi, S.; Sakamoto, H.; Nakanishi, Y.; Yamanishi, M.; Mori, K.; Toraya, T.
Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli
J. Biochem.
147
83-93
2010
Escherichia coli
Sagermann, M.; Ohtaki, A.; Nikolakakis, K.
Crystal structure of the EutL shell protein of the ethanolamine ammonia lyase microcompartment
Proc. Natl. Acad. Sci. USA
106
8883-8887
2009
Escherichia coli
Shibata, N.; Tamagaki, H.; Ohtsuki, S.; Hieda, N.; Akita, K.; Komori, H.; Shomura, Y.; Terawaki, S.; Toraya, T.; Yasuoka, N.; Higuchi, Y.
Expression, crystallization and preliminary X-ray crystallographic study of ethanolamine ammonia-lyase from Escherichia coli
Acta Crystallogr. Sect. F
66
709-711
2010
Escherichia coli (P19636), Escherichia coli
Shibata, N.; Higuchi, Y.; Toraya, T.
How coenzyme B12-dependent ethanolamine ammonia-lyase deals with both enantiomers of 2-amino-1-propanol as substrates: structure-based rationalization
Biochemistry
50
591-598
2011
Escherichia coli (P19636)
Shibata, N.; Tamagaki, H.; Hieda, N.; Akita, K.; Komori, H.; Shomura, Y.; Terawaki, S.; Mori, K.; Yasuoka, N.; Higuchi, Y.; Toraya, T.
Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates
J. Biol. Chem.
285
26484-26493
2010
Escherichia coli (P19636), Escherichia coli
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