Literature summary for 2.3.1.31 extracted from

Zubieta, C.; Arkus, K.A.; Cahoon, R.E.; Jez, J.M.
A single amino acid change is responsible for evolution of acyltransferase specificity in bacterial methionine biosynthesis (2008), J. Biol. Chem., 283, 7561-7567.

Cloned(Commentary)

Cloned (Comment)	Organism
amplification, expression in Escherichia coli BL21(DE3)	Bacillus cereus
expressed in Escherichia coli as a His-tagged fusion protein	Bacillus cereus

Crystallization (Commentary)

Crystallization (Comment)	Organism
enzyme in complex with homoserine, hanging drop vapor diffusion, purified protein is added to 1.4 M (NH4)2SO4 and 0.1 M Tris, pH 8.0, soaked with 1.8 M (NH4)2SO4 and same buffer containing 15% glycerol, and 10 mM homoserine, crystals are flash frozen	Bacillus cereus
x-ray crystal structure at 2.0 A of the Bacillus cereus metA protein in complex with homoserine is presented	Bacillus cereus

Protein Variants

Protein Variants	Comment	Organism
C142A	inactive enzyme	Bacillus cereus
C142A	enzyme inactive	Bacillus cereus
C142S	inactive enzyme	Bacillus cereus
C142S	enzyme inactive	Bacillus cereus
E111G	mutant protein shows no detectable activity with acetyl-CoA but catalyzes an acyltransferase reaction using succinyl-CoA and homoserine (kcat (succinyl-CoA): 0.8/sec, Km (succinyl-CoA): 0.273 mM, Km (L-homoserine): 0.2 mM)	Bacillus cereus
E111G	no activity with acetyl-CoA, but with succinyl-CoA and homoserine, glutamic acid 111 (corresponding to Escherichia coli residue with function in succinyl-specificity of homoserine transsuccinylase) sterically occludes fitting of a succinyl-enzyme intermediate in the active site	Bacillus cereus
E237A	decrease in catalytic activity	Bacillus cereus
E237A	compared to wild-type: kcat and Km (acetyl-CoA) decreased, Km (L-homoserine) increased	Bacillus cereus
E237D	decrease in catalytic activity	Bacillus cereus
E237D	compared to wild-type: kcat and Km (acetyl-CoA) decreased, Km (L-homoserine) increased	Bacillus cereus
E237Q	decrease in catalytic activity	Bacillus cereus
E237Q	compared to wild-type: kcat and Km (acetyl-CoA) decreased, Km (L-homoserine) increased	Bacillus cereus
E250A	compared to wild-type: kcat and Km (acetyl-CoA) decreased, Km (L-homoserine) increased	Bacillus cereus
E250A	13-14fold increase in homoserine Km value, homoserine binding is affected not acetyl-CoA binding	Bacillus cereus
H235A	inactive enzyme	Bacillus cereus
H235A	enzyme inactive	Bacillus cereus
H235N	inactive enzyme	Bacillus cereus
H235N	enzyme inactive	Bacillus cereus
H235Q	inactive enzyme	Bacillus cereus
H235Q	enzyme inactive	Bacillus cereus
K163M	compared to wild-type: kcat (acetyl-CoA) decreased, Km (L-homoserine) and (acetyl-CoA) increased	Bacillus cereus
K163M	13-14fold increase in homoserine Km value, homoserine binding is affected not acetyl-CoA binding	Bacillus cereus
K47M	compared to wild-type: kcat (acetyl-CoA) decreased, Km (L-homoserine) and (acetyl-CoA) increased	Bacillus cereus
K47M	turnover number is reduced 14fold, Km value for acetyl-CoA is reduced 17fold, function in acetyl-CoA binding	Bacillus cereus
K47R	compared to wild-type: kcat (acetyl-CoA) decreased, Km (L-homoserine) and (acetyl-CoA) increased	Bacillus cereus
K47R	Km value for acetyl-CoA is affected, function in acetyl-CoA binding	Bacillus cereus
R249M	compared to wild-type: kcat (acetyl-CoA) decreased, Km (L-homoserine) and (acetyl-CoA) increased	Bacillus cereus
R249M	10fold reduction in kcat, 64fold higher Km for homoserine than wild-type, homoserine binding is affected not acetyl-CoA binding	Bacillus cereus
S192A	compared to wild-type: kcat (acetyl-CoA) decreased, Km (L-homoserine) and (acetyl-CoA) increased	Bacillus cereus
S192A	5fold increase in Km for homoserine, homoserine binding is affected not acetyl-CoA binding	Bacillus cereus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.108	-	acetyl-CoA	mutant E237A	Bacillus cereus
0.108	-	acetyl-CoA	E237A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.143	-	acetyl-CoA	mutant E237Q	Bacillus cereus
0.143	-	acetyl-CoA	E237Q, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.165	-	acetyl-CoA	mutant E250A	Bacillus cereus
0.165	-	acetyl-CoA	E250A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.174	-	acetyl-CoA	mutant E237D	Bacillus cereus
0.174	-	acetyl-CoA	E237D, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.185	-	acetyl-CoA	wild-type	Bacillus cereus
0.185	-	acetyl-CoA	wild-type, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.199	-	acetyl-CoA	mutant R249M	Bacillus cereus
0.199	-	acetyl-CoA	R249M , 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.2	-	L-homoserine	E111G, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM succinyl-CoA, 25°C	Bacillus cereus
0.214	-	L-homoserine	wild-type	Bacillus cereus
0.214	-	L-homoserine	wild-type, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.249	-	acetyl-CoA	mutant S192A	Bacillus cereus
0.249	-	acetyl-CoA	S192A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.266	-	acetyl-CoA	mutant K163M	Bacillus cereus
0.266	-	acetyl-CoA	K163M, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.273	-	succinyl-CoA	E111G, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.287	-	L-homoserine	mutant E237Q	Bacillus cereus
0.287	-	L-homoserine	E237Q, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.405	-	L-homoserine	mutant E237D	Bacillus cereus
0.405	-	L-homoserine	E237D, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.85	-	L-homoserine	mutant E237A	Bacillus cereus
0.85	-	L-homoserine	E237A, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.946	-	L-homoserine	mutant K47R	Bacillus cereus
0.946	-	L-homoserine	K47R, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
1.02	-	L-homoserine	mutant S192A	Bacillus cereus
1.02	-	L-homoserine	S192A, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
1.35	-	acetyl-CoA	mutant K47R	Bacillus cereus
1.35	-	acetyl-CoA	K47R, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
1.38	-	L-homoserine	mutant K47M	Bacillus cereus
1.38	-	L-homoserine	K47M, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
2.78	-	L-homoserine	mutant K163M	Bacillus cereus
2.78	-	L-homoserine	K163M, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
2.95	-	L-homoserine	mutant E250A	Bacillus cereus
2.95	-	L-homoserine	E250A, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
3.07	-	acetyl-CoA	mutant K47M	Bacillus cereus
3.07	-	acetyl-CoA	K47M, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
13.8	-	L-homoserine	mutant R249M	Bacillus cereus
13.8	-	L-homoserine	R249M, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
acetyl-CoA + L-homoserine	Bacillus cereus	functions as homoserine transacetylase rather than homoserine transsuccinylase despite its more than 50% sequence identity with homoserine transsuccinylase	CoA + O-acetyl-L-homoserine	-	?
succinyl-CoA + L-homoserine	Bacillus cereus	activity of mutant E111G	CoA + O-succinyl-L-homoserine	-	?

Organism

Organism	UniProt	Comment	Textmining
Bacillus cereus	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
cells are centrifuged in 50 mM Tris, pH 8.0, sonicated, centrifuged, supernatant loaded to a Ni2+-nitrilotriacetic acid-agarose column, washed, and eluted, eland incubated with protease and dialyzed, reloaded onto a Ni2+-nitrilotriacetic acid-agarose column, dialyzed, loaded onto Superdex-200 size-exclusion fast protein liquid chromatography column with 25 mM HEPES-buffer, pH 7.5	Bacillus cereus
using Ni-NTA-chromatography	Bacillus cereus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
acetyl-CoA + L-homoserine	-	Bacillus cereus	CoA + O-acetyl-L-homoserine	-	?
acetyl-CoA + L-homoserine	functions as homoserine transacetylase rather than homoserine transsuccinylase despite its more than 50% sequence identity with homoserine transsuccinylase	Bacillus cereus	CoA + O-acetyl-L-homoserine	-	?
succinyl-CoA + L-homoserine	activity of mutant E111G	Bacillus cereus	CoA + O-succinyl-L-homoserine	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * x, crystal structure with substrate L-homoserine	Bacillus cereus

Synonyms

Synonyms	Comment	Organism
homoserine O-acetyltransferase	-	Bacillus cereus
homoserine transsuccinylase	authors state, in reality it is a homoserine transacetylase	Bacillus cereus
HTA	-	Bacillus cereus
HTS	authors state, in reality it is a HTA	Bacillus cereus
MetA	-	Bacillus cereus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Bacillus cereus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.015	-	acetyl-CoA	E237A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.02	-	acetyl-CoA	mutant E237A	Bacillus cereus
0.05	-	acetyl-CoA	mutant E237Q	Bacillus cereus
0.052	-	acetyl-CoA	E237Q, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.07	-	acetyl-CoA	mutant K47M	Bacillus cereus
0.07	-	acetyl-CoA	K47M, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.1	-	acetyl-CoA	mutant R249M	Bacillus cereus
0.101	-	acetyl-CoA	R249M , 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.158	-	acetyl-CoA	E237D, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.16	-	acetyl-CoA	mutant E237D	Bacillus cereus
0.328	-	acetyl-CoA	K163M, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.33	-	acetyl-CoA	mutant K163M	Bacillus cereus
0.435	-	acetyl-CoA	E250A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.44	-	acetyl-CoA	mutant E250A	Bacillus cereus
0.54	-	acetyl-CoA	mutant S192A	Bacillus cereus
0.543	-	acetyl-CoA	S192A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.655	-	acetyl-CoA	K47R, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.66	-	acetyl-CoA	mutant K47R	Bacillus cereus
0.803	-	succinyl-CoA	E111G, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.977	-	acetyl-CoA	wild-type, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.98	-	acetyl-CoA	wild-type	Bacillus cereus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Bacillus cereus

General Information

General Information	Comment	Organism
malfunction	site-directed mutagenesis reveals that Bacillus cereus metA and Escherichia coli homoserine transsuccinylase share a common catalytic mechanism, glutamic acid 111 in the active site determines acetyl-CoA versus succinyl-CoA (glycine 111) specificity	Bacillus cereus
metabolism	methionine biosynthesis	Bacillus cereus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.0074	-	L-homoserine	R249M, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.0176	-	L-homoserine	E237A, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.0228	-	acetyl-CoA	K47M, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.0507	-	L-homoserine	K47M, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.118	-	L-homoserine	K163M, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.139	-	acetyl-CoA	E237A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.147	-	L-homoserine	E250A, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.18	-	L-homoserine	E237Q, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.361	-	acetyl-CoA	E237Q, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.391	-	L-homoserine	E237D, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.485	-	acetyl-CoA	K47R, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.511	-	acetyl-CoA	R249M , 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
0.533	-	L-homoserine	S192A, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.692	-	L-homoserine	K47R, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
0.91	-	acetyl-CoA	E237D, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
1.23	-	acetyl-CoA	K163M, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
2.18	-	acetyl-CoA	S192A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
2.64	-	acetyl-CoA	E250A, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
2.94	-	succinyl-CoA	E111G, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus
4.02	-	L-homoserine	E111G, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM succinyl-CoA, 25°C	Bacillus cereus
4.56	-	L-homoserine	wild-type, 50 mM potassium phosphate buffer, pH 7.5, 0.5 mM acetyl-CoA, 25°C	Bacillus cereus
5.28	-	acetyl-CoA	wild-type, 50 mM potassium phosphate buffer, pH 7.5, 2 mM L-homoserine, 25°C	Bacillus cereus