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 (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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus cereus | - |
- |
- |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Comment | Organism |
---|---|---|
homodimer | 2 * x, crystal structure with substrate L-homoserine | Bacillus cereus |
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 Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Bacillus cereus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Bacillus cereus |
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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 |