Information on EC 2.3.1.46 - homoserine O-succinyltransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.3.1.46
-
RECOMMENDED NAME
GeneOntology No.
homoserine O-succinyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
succinyl-CoA + L-homoserine = CoA + O-succinyl-L-homoserine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
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Cysteine and methionine metabolism
-
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L-methionine biosynthesis I
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Metabolic pathways
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Sulfur metabolism
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methionine metabolism
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SYSTEMATIC NAME
IUBMB Comments
succinyl-CoA:L-homoserine O-succinyltransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
62213-51-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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UniProt
Manually annotated by BRENDA team
strain W3110, gene metA
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Manually annotated by BRENDA team
KCTC 3397
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
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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
additional information
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the enzyme is prone to aggregation under many stress conditions, resulting in a methionine limitation in Escherichia coli growth
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
glutaryl-CoA + L-homoserine
CoA + O-glutaryl-L-homoserine
show the reaction diagram
-
-
-
-
r
succinyl-CoA + D-homoserine
CoA + O-succinyl-D-homoserine
show the reaction diagram
-
-
-
-
r
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
D-methionine
diethyldicarbonate
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inactivation, pH dependence, overview
Hydroxyethylclavam
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oxygen analogue beta-lactam antibiotic
iodoacetamide
L-methionine
S-adenosyl-L-methionine
Valclavam
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oxygen analogue beta-lactam antibiotic, complete inhibition at 0.2 mM
additional information
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overview, competitive antagonism of peptides against enzyme inhibition by valclavam; overview, inhibition of several fungi and procaryotes by valclavam
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.64
coenzyme A
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recombinant protein
10
D-homoserine
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recombinant protein
0.18
glutaryl-CoA
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recombinant protein
0.0032 - 95.5
L-homoserine
3.5
O-succinylhomoserine
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recombinant protein
0.043 - 2.9
succinyl-CoA
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.23
coenzyme A
Escherichia coli
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recombinant protein
12
D-homoserine
Escherichia coli
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recombinant protein
1.6
glutaryl-CoA
Escherichia coli
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recombinant protein
0.034 - 130
L-homoserine
5.23
O-succinyl-L-homoserine
Escherichia coli
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recombinant protein
0.34 - 130
succinyl-CoA
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
27.6 - 46.5
L-homoserine
345
99 - 109
succinyl-CoA
224
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00025 - 0.00092
hydroxyethylvalclavam
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0.00083 - 0.00089
Valclavam
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
500
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wild-type histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, about 80% less activity as at 25°C
520
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mutant N267D histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, almost 5times higher activity at 25°C
610
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mutant I229T histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, about 4times higher activity at 25°C
700
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histidine-tagged enzyme, 50 mM potassium-phosphate buffer, pH 7.5, 40°C, about 3.5times higher activity as at 25°C
1100
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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
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
additional information
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broad maximum
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4 - 7.5
6 - 8.7
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6.2 - 8.5
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pH profile of mutant K47R
6.9 - 8
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crude extract, about 40% of maximal activity at pH 6.9, about 80% of maximal activity at pH 8.0
additional information
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pKs of 6.6 and about 7.9
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 58
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus cereus (strain ATCC 10987 / NRS 248)
Bacillus cereus (strain ATCC 10987 / NRS 248)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35600
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2 * 35600, SDS-PAGE
70000
gel filtration
86000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of HTS from Bacillus cereus is determined to 2.4 A resolution. HTS is a single-domain protein with a Rossmann fold topology. The core of the protein is a parallel beta-sheet sandwiched by alpha-helices. HTS is composed of 11 beta-strands, 7 alpha-helices, and four 310-helices
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
44
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complete aggregation of wild-tyype enzyyme at 44°C
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
glycerol, 30% w/v, stabilizes during purification
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inherently unstable and prone to aggregation, the enzyme is prone to aggregation under many stress conditions, resulting in a methionine limitation in Escherichia coli growth
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
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recombinant from E. coli BL21 (DE3)
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recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by 2 different steps of anion exchange chromatography
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recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange chromatography, ammonium sulfate fractionation, hydrophobic interaction chromatography, and dialysis to over 90% purity
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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
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
gene metA, expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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gene metA, expressionof wild-type and mutant enzymes in strain JM109
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gene metA, recombinant expression of C-terminally His-tagged wild-type and mutant enzymes in enzyme-deficient Escherichia coli strain JW3973
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Geobacillus kaustophilus gene is transferred into Escherichia coli JW3973
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metA gene, expression in Escherichia coli auxotrophic strains as lacZ fusion proteins
metA, expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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overexpression of metA gene in Escherichia coli BL21 (DE3)
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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)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A195T
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site-directed mutagenesis
A200E
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site-directed mutagenesis
C142A
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site-directed mutagenesis, the mutant is inactive
C90A
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site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme
C90S
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site-directed mutagenesis, the mutant shows only slightly reduced activity compared to the wild-type enzyme
D218G
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site-directed mutagenesis
E213V
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15-20% faster growth at 36-41°C, lower growth rate at 44°C (64% of control), no growth at 45°C
E237A
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
E237D
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E246A
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
E246D
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site-directed mutagenesis, the mutant shows reduced catalytic efficiency with L-homoserine, but increased with succinyl-CoA compared to the wild-type enzyme
F247Y
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site-directed mutagenesis, the mutant shows accelerated growth at 44°C in M9 glucose medium in contrast to the wild-type
H235A
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site-directed mutagenesis, the mutant is inactive
I124L
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site-directed mutagenesis, the mutant shows accelerated growth at 44°C in M9 glucose medium in contrast to the wild-type
I124L-I229Y
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site-directed mutagenesis, the mutant shows highly accelerated growth at 44°C in M9 glucose medium in contrast to the wild-type
I124L-I229Y-N267D
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site-directed mutagenesis, the mutant shows highly accelerated growth at 44°C in M9 glucose medium in contrast to the wild-type
I229T
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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
I296S
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A4
I299Y
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site-directed mutagenesis, the mutant shows accelerated growth at 44°C in M9 glucose medium in contrast to the wild-type
K156L
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site-directed mutagenesis, the mutant is inactive
K45A/K46A
K45L
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
K46A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
K46L
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
K46R
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
K46R/K47R
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
K47A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
K47R
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site-directed mutagenesis, the mutant shows 90% reduced activity compared to the wild-type enzyme
L110V
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site-directed mutagenesis
N267D
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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
N271K
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15-20% faster growth at 36-41°C, lower growth rate at 44°C (22% of control), no growth at 45°C
P298L
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A5
Q96K
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site-directed mutagenesis, the mutant shows accelerated growth at 44°C in M9 glucose medium in contrast to the wild-type
R160L
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site-directed mutagenesis
R193A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
R193A/E246A
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site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency with L-homoserine and reduced activity with succinyl-CoA compared to the wild-type enzyme
R193K
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
R249A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
R249K
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
R27C
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A9
S61T
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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
S61T/E213V/I229T/N267D/N271K
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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
Y238F
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Y238F/E246A
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site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency with L-homoserine, but increased with succinyl-CoA compared to the wild-type enzyme
I296S
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A4
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P298L
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A5
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R27C
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A9
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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