Cloned (Comment) | Organism |
---|---|
Geobacillus kaustophilus gene is transferred into Escherichia coli JW3973 | Geobacillus kaustophilus |
PCR-amplification of mutant Escherichia coli genes, transfer into null mutant Escherichia coli JW3973, thermoselection at 44°C, and mutated or wild-type gene metA transformed into Escherichia coli BL21(DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
E213V | 15-20% faster growth at 36-41°C, lower growth rate at 44°C (64% of control), no growth at 45°C | Escherichia coli |
I229T | normal growth at 37°C, better growth at 44°C compared to control, 48% faster growth at 44°C, 18% faster growth at 43°C, no growth at 45°C, 1.4times greater cell density with 30 mM acetic acid at 30°C than control, heating at 45°C for 40 min reduces soluble enzyme to 29% compared to unheated culture, increases insoluble enzyme content 13-19times, 3 h incubation in 30 mM acetic acid at 30°C shows 1.5-3times higher levels of soluble enzyme than the wild-type strain and 2.6times higher insoluble enzyme levels | Escherichia coli |
additional information | the recombinant Geobacillus kaustophilus enzyme stimulates Escherichia coli growth at 44°C (20% faster than control) but inhibits growth by 20% at 37°C compared to the wild-type, between 30-42°C growth is inhibited by about 10%, at 43°C growth is equal to the Escherichia coli wild-type enzyme | Geobacillus kaustophilus |
additional information | wild-type Escherichia coli enzyme, normal growth at 37°C, less growth than the mutants at 44°C no growth at 45°C, heating at 45°C for 40 min reduces soluble enzyme to 35% compared to unheated culture, increases insoluble enzyme content 44times, 3 h incubation in 30 mM acetic acid at 30°C shows 1.5-3times lower levels of soluble enzyme than the mutants and 15times higher insoluble enzyme levels | Escherichia coli |
N267D | normal growth at 37°C, better growth at 44°C compared to control, 31% faster growth at 44°C, 10% faster growth at 43°C, no growth at 45°C, with 30 mM acetic acid at 30°C slower growth than other thermostable strains and only 16% higher cell density than control, heating at 45°C for 40 min reduces soluble enzyme to 67% compared to unheated culture, increases insoluble enzyme content 13-19times, 3 h incubation in 30 mM acetic acid at 30°C shows 1.5-3times higher levels of soluble enzyme than the wild-type strain and 8times higher insoluble enzyme levels | Escherichia coli |
N271K | 15-20% faster growth at 36-41°C, lower growth rate at 44°C (22% of control), no growth at 45°C | Escherichia coli |
S61T | similar growth rate as mutants E213V and N271K at 36-41°C (15-20% faster), similar growth as wild-type at elevated temperatures, no growth at 45°C | Escherichia coli |
S61T/E213V/I229T/N267D/N271K | introduction of random mutagenesis by error-prone PCR, fast growing strain at 44°C, 5-12% faster growing than control strain at a temperature range from 30-42°C, at 43°C 30% faster growth, at 44°C 64% faster growth, no growth at 45°C, 1.4fold slower growth in methionine deficient medium at 43 and 44°C compared to 1.6 and 2fold slowing in the wild-type, 1.4times greater cell density with 30 mM acetic acid at 30°C than control, heating at 45°C for 40 min reduces soluble enzyme to 58% compared to unheated culture, increases insoluble enzyme content 13-19times, 3 h incubation in 30 mM acetic acid at 30°C shows 1.5-3times higher levels of soluble enzyme than the wild-type strain and 9times higher insoluble enzyme levels | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
succinyl-CoA + L-homoserine | Escherichia coli | - |
CoA + O-succinyl-L-homoserine | - |
? | |
succinyl-CoA + L-homoserine | Geobacillus kaustophilus | - |
CoA + O-succinyl-L-homoserine | - |
? | |
succinyl-CoA + L-homoserine | Geobacillus kaustophilus KCTC 3397 | - |
CoA + O-succinyl-L-homoserine | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
wild-type W3110, and JW3973 (homoserine O-succinyltransferase null mutant) | - |
Geobacillus kaustophilus | - |
KCTC 3397 | - |
Geobacillus kaustophilus KCTC 3397 | - |
KCTC 3397 | - |
Purification (Comment) | Organism |
---|---|
Escherichia coli cells are lysed with 0.5 mg/ml lysozyme-1 mM phenylmethylsulfonyl fluoride-DNase I, sonicated, centrifuged, supernatants purified with Ni-nitrilo-triacetic acid agarose, gravity filtration of unbound protein, elution with 50 mM Tris-HCl, pH 7.5, NaCl, and imidazole, dialysis with potassium-phosphate buffer, pH 7.6 | Escherichia coli |
transformed Escherichia coli cells are lysed with 0.5 mg/ml lysozyme-1 mM phenylmethylsulfonyl fluoride-DNase I and sonicated, centrifuged, supernatants purified with Ni-nitrilo-triacetic acid agarose, gravity filtration of unbound protein, elution with 50 mM Tris-HCl, pH 7.5, NaCl, and imidazole, dialysis with potassium-phosphate buffer, pH 7.6 | Geobacillus kaustophilus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
500 | - |
wild-type histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, about 80% less activity as at 25°C | Escherichia coli |
520 | - |
mutant N267D histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, almost 5times higher activity at 25°C | Escherichia coli |
610 | - |
mutant I229T histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, about 4times higher activity at 25°C | Escherichia coli |
700 | - |
histidine-tagged enzyme, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, about 3.5times higher activity as at 25°C | Geobacillus kaustophilus |
1100 | - |
mutant 333 histidine-tagged (S61T/E213V/I229T/N267D/N271K), 50 mM potassium-phosphate buffer, pH 7.5, 40°C, about 50% less active as at 25°C | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
succinyl-CoA + L-homoserine | - |
Escherichia coli | CoA + O-succinyl-L-homoserine | - |
? | |
succinyl-CoA + L-homoserine | - |
Geobacillus kaustophilus | CoA + O-succinyl-L-homoserine | - |
? | |
succinyl-CoA + L-homoserine | - |
Geobacillus kaustophilus KCTC 3397 | CoA + O-succinyl-L-homoserine | - |
? |
Synonyms | Comment | Organism |
---|---|---|
homoserine O-succinyltransferase | - |
Escherichia coli |
homoserine O-succinyltransferase | - |
Geobacillus kaustophilus |
MetA protein | - |
Escherichia coli |
MetA protein | - |
Geobacillus kaustophilus |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | 58 | pH 7.5, histidine-tagged enzymes, the mutant strain 333 (S61T/E213V/I229T/N267D/N271K) enzyme is about 2.5times more active than the wild-type between 40-55°C but has no activity at 58°C, mutants N267D and I229T show lower activity from 40-45°C and similar activity profiles at 50°C and beyond | Escherichia coli |
40 | 58 | pH 7.5, the recombinant enzyme shows a decrease in activity with temperature but still activity at 58°C at about 180 microM/min/mg protein | Geobacillus kaustophilus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
recombinant enzyme shows high transition temperature of 67.68°C | Geobacillus kaustophilus |
additional information | - |
the transition temperature of wild-type enzyme is 47.075°C, of the mutant strain 333 (S61T/E213V/I229T/N267D/N271K) 48.76°C, of mutant N267D 47.36°C, and of mutant I229T 46.99°C | Escherichia coli |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5.4 | 7.5 | 25°C, histidine-tagged enzyme, 0-50 mM acetic acid: 0 mM = pH 7.5, 10 mM = 7.0, 20 mM = 6.6, 30 mM = 6.2, 40 mM = 5.8, 50 mM = 5.4, the recombinant enzyme shows reduced activity at 25°C without great change at about 200 micromol/min/mg protein, about 3.5times higher activity at 40°C | Geobacillus kaustophilus |
5.4 | 7.5 | 25°C, histidine-tagged enzymes, 0-50 mM acetic acid: 0 mM = pH 7.5, 10 mM = 7.0, 20 mM = 6.6, 30 mM = 6.2, 40 mM = 5.8, 50 mM = 5.4, sharp decrease of wild-type enzyme activity above 30 mM acetic acid, no activity at 50 mM, without acid about 2900 micromol/min/mg protein, the mutant strain 333 (S61T/E213V/I229T/N267D/N271K) enzyme retains almost 50% activity at 50 mM acetic acid, activity decreases above 30 mM from about 2000 micromol/min/mg protein, the mutants N267D and I229T do not greatly change their activity, about 2000-1800 micromol/min/mg protein, and about 2500-2000 micromol/min/mg protein, respectively | Escherichia coli |
General Information | Comment | Organism |
---|---|---|
metabolism | first enzyme in the methionine biosynthesis pathway | Escherichia coli |
metabolism | first enzyme in the methionine biosynthesis pathway | Geobacillus kaustophilus |
physiological function | methionine (end product of enzyme) accelerates Escherichia coli W3110 growth at temperatures above 30°C and enables growth at 45 and 46°C, without methionine slow growth at 45°C and no growth at 46°C, no growth at 47°C and beyond even with methionine | Escherichia coli |