Literature summary for 4.3.1.7 extracted from

Sun, L.; Groover, O.A.; Canfield, J.M.; Warncke, K.
Critical role of arginine 160 of the EutB protein subunit for active site structure and radical catalysis in coenzyme B12-dependent ethanolamine ammonia-lyase (2008), Biochemistry, 47, 5523-5535.

Cloned(Commentary)

Cloned (Comment)	Organism
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.	Salmonella enterica subsp. enterica serovar Typhimurium

Cloned (Comment)

Organism

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.

Salmonella enterica subsp. enterica serovar Typhimurium

Protein Variants

Protein Variants	Comment	Organism
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.	Salmonella enterica subsp. enterica serovar Typhimurium
R160E	mutant fails to assemble into an oligomer	Salmonella enterica subsp. enterica serovar Typhimurium
R160I	mutant fails to assemble into an oligomer	Salmonella enterica subsp. enterica serovar Typhimurium
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	Salmonella enterica subsp. enterica serovar Typhimurium

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.0136	-	ethanolamine	wild-type	Salmonella enterica subsp. enterica serovar Typhimurium
0.41	-	ethanolamine	R160K EAL shows a 30fold increase relative to wild-type enzyme	Salmonella enterica subsp. enterica serovar Typhimurium

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
32100	-	eutC protein subunit contains 286 amino acids	Salmonella enterica subsp. enterica serovar Typhimurium
49400	-	eutB protein subunit contains 453 amino acids and the active site	Salmonella enterica subsp. enterica serovar Typhimurium
500000	-	whole enzyme, indicates that the two subunits are present in a EutB6EutC6 stoichiometry	Salmonella enterica subsp. enterica serovar Typhimurium

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ethanolamine	Salmonella enterica subsp. enterica serovar Typhimurium	-	acetaldehyde + NH3	-	?

Organism

Organism	UniProt	Comment	Textmining
Salmonella enterica subsp. enterica serovar Typhimurium	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Salmonella enterica subsp. enterica serovar Typhimurium

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	Externally added guanidine hydrochloride leads to significant R160A EAL enzyme activity under Vmax conditions, the activity of R160A EAL increases with guanidine hydrochloride concentration and reaches a maximum at 10 mM, which represents 2.3% of the activity of the wild-type control. The resurrection of enzyme activity in R160A EAL by externally added guanidine hydrochloride provides strong support that a positively charged arginine side chain in the active site is catalytically essential.	Salmonella enterica subsp. enterica serovar Typhimurium

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
(S)-2-aminopropanol	-	Salmonella enterica subsp. enterica serovar Typhimurium	?	-	?
ethanolamine	-	Salmonella enterica subsp. enterica serovar Typhimurium	acetaldehyde + NH3	-	?

Subunits

Subunits	Comment	Organism
oligomer	Two subunits are present in a EutB6EutC6 stoichiometry, EAL is composed of a 453-residue EutB protein subunit and a 286-residue EutC protein subunit, which are coded by the eutb and eutc genes. The positive charge at the terminus of the R160 side chain of EutB is required to achieve the native EutB protein fold and the oligomeric structure of EAL	Salmonella enterica subsp. enterica serovar Typhimurium

Synonyms

Synonyms	Comment	Organism
AEL	-	Salmonella enterica subsp. enterica serovar Typhimurium
ethanolamine ammonia-lyase	-	Salmonella enterica subsp. enterica serovar Typhimurium
ethanolamine deaminase	-	Salmonella enterica subsp. enterica serovar Typhimurium
EutB	-	Salmonella enterica subsp. enterica serovar Typhimurium

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
additional information	-	additional information	mutant R160A displays no detectable catalytic turnover with ethanolamine as subtrate	Salmonella enterica subsp. enterica serovar Typhimurium
8.5	-	ethanolamine	R160K EAL	Salmonella enterica subsp. enterica serovar Typhimurium
52.2	-	ethanolamine	wild-type	Salmonella enterica subsp. enterica serovar Typhimurium

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Salmonella enterica subsp. enterica serovar Typhimurium

Cofactor

Cofactor	Comment	Organism	Structure
coenzyme B12	adenosylcobalamin	Salmonella enterica subsp. enterica serovar Typhimurium