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Literature summary for 2.3.1.46 extracted from

  • Mordukhova, E.A.; Lee, H.S.; Pan, J.G.
    Improved thermostability and acetic acid tolerance of Escherichia coli via directed evolution of homoserine o-succinyltransferase (2008), Appl. Environ. Microbiol., 74, 7660-7668.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.3.1.46

Cloned(Commentary)

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

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/ Products (Substrates)

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

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 (Commentary)

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 [micromol/min/mg]

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 and Products (Substrate)

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

Synonyms Comment Organism
homoserine O-succinyltransferase
-
 Escherichia coli
homoserine O-succinyltransferase
-
 Geobacillus kaustophilus
MetA protein
-
 Escherichia coli
MetA protein
-
 Geobacillus kaustophilus

Temperature Range [°C]

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 [°C]

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 Range

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

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