Information on EC 2.3.1.46 - homoserine O-succinyltransferase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY
2.3.1.46
-
RECOMMENDED NAME
GeneOntology No.
homoserine O-succinyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
succinyl-CoA + L-homoserine = CoA + O-succinyl-L-homoserine
show the reaction diagram
ping-pong mechanism, enzyme contains a cysteine in the active site, member of the acyltransferase family
-
succinyl-CoA + L-homoserine = CoA + O-succinyl-L-homoserine
show the reaction diagram
succinyl is covalently bound to one of two adjacent lysine residues at positions 45 and 46
-
succinyl-CoA + L-homoserine = CoA + O-succinyl-L-homoserine
show the reaction diagram
ping pong catalytic reaction mechanism, roles of essential functional groups, the catalytic triad is formed by the nucleophile Cys142, the essential base His235, and Glu237, required for proper orientation of His235, Lys46 is required for interaction with succinyl-CoA, and Arg193 is involved in binding of L-Homoserine
-
succinyl-CoA + L-homoserine = CoA + O-succinyl-L-homoserine
show the reaction diagram
ping pong catalytic reaction mechanism, the residues Cys142, His235, and Lys47 are essential for catalytic activity, Cys142 acts as nucleophile to which the succinyl moiety is transferred during the reaction followed by transfer to homoserine to form O-succinylhomoserine, Lys47 is involved in the first half-reaction, and His235 is the active site base
-
succinyl-CoA + L-homoserine = CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Acyl group transfer
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
Cysteine and methionine metabolism
-
Metabolic pathways
-
methionine biosynthesis I
-
Sulfur metabolism
-
SYSTEMATIC NAME
IUBMB Comments
succinyl-CoA:L-homoserine O-succinyltransferase
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
homoserine O-succinyltransferase
Q72X44
-
homoserine O-succinyltransferase
-
-
homoserine O-succinyltransferase
Geobacillus kaustophilus KCTC 3397
-
-
-
homoserine O-transsuccinylase
-
-
-
-
homoserine succinyltransferase
-
-
-
-
homoserine succinyltransferase
-
-
homoserine succinyltransferase
Escherichia coli W3110
-
-
-
homoserine transsuccinylase
-
-
HTS
Q72X44
-
MetA
Escherichia coli W3110
-
-
-
MetA protein
Geobacillus kaustophilus KCTC 3397
-
-
-
succinyltransferase, homoserine
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
62213-51-8
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
auxotrophic mutants derived from K-12; metA gene; strain K-12
-
-
Manually annotated by BRENDA team
auxotrophic strains
-
-
Manually annotated by BRENDA team
gene metA
-
-
Manually annotated by BRENDA team
metA gene
-
-
Manually annotated by BRENDA team
strain K-12
-
-
Manually annotated by BRENDA team
strain W3110, gene metA
-
-
Manually annotated by BRENDA team
wild-type W3110, and JW3973 (homoserine O-succinyltransferase null mutant)
-
-
Manually annotated by BRENDA team
Escherichia coli W3110
strain W3110, gene metA
-
-
Manually annotated by BRENDA team
Geobacillus kaustophilus KCTC 3397
KCTC 3397
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
metabolism
-
first enzyme in the methionine biosynthesis pathway
metabolism
Geobacillus kaustophilus KCTC 3397
-
first enzyme in the methionine biosynthesis pathway
-
physiological function
-
methionine (end product of enzyme) accelerates Escherichia coli W3110 growth at temperatures above 30C and enables growth at 45 and 46C, without methionine slow growth at 45C and no growth at 46C, no growth at 47C and beyond even with methionine
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
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
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
r
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
involved in cystathionine and methionine biosynthesis
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
involved in cystathionine and methionine biosynthesis
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
involved in cystathionine and methionine biosynthesis
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
a step in biosynthesis and regulation of methionine, overview
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
step in the biosynthetic pathways of methionine and S-adenosylmethionine, overview
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
Geobacillus kaustophilus KCTC 3397
-
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
Escherichia coli W3110
-
-, a step in biosynthesis and regulation of methionine, overview
-
-
?
glutaryl-CoA + L-homoserine
CoA + O-glutaryl-L-homoserine
show the reaction diagram
-
-
-
-
r
additional information
?
-
-
3-amino-1-propanol, L-serine, and L-threonine are poor substrates, no activity with acetyl-CoA, propionyl-CoA, butyryl-CoA, crotonyl-CoA, malonyl-CoA, and gamma-hydroxybutyric acid, and gamma-aminobutyric acid
-
-
-
additional information
?
-
Escherichia coli, Escherichia coli W3110
-
the enzyme is important in regulation of Met biosynthesis and metabolism, metA expression is regulated by the methionine repressor metJ, MetJ and MetA enzyme mutations lead to pathway deregulation and excess methionine production and excretion from the cell, overview
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
involved in cystathionine and methionine biosynthesis
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
involved in cystathionine and methionine biosynthesis
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
involved in cystathionine and methionine biosynthesis
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
a step in biosynthesis and regulation of methionine, overview
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
-
step in the biosynthetic pathways of methionine and S-adenosylmethionine, overview
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
Geobacillus kaustophilus KCTC 3397
-
-
-
-
?
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
Escherichia coli W3110
-
a step in biosynthesis and regulation of methionine, overview
-
-
?
additional information
?
-
Escherichia coli, Escherichia coli W3110
-
the enzyme is important in regulation of Met biosynthesis and metabolism, metA expression is regulated by the methionine repressor metJ, MetJ and MetA enzyme mutations lead to pathway deregulation and excess methionine production and excretion from the cell, overview
-
-
-
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
diethyldicarbonate
-
inactivation, pH dependence, overview
Hydroxyethylclavam
-
oxygen analogue beta-lactam antibiotic
iodoacetamide
-
inhibition is pH-dependent
iodoacetamide
-
inactivation, pH dependence, overview
L-methionine
-
feed-back inhibition
S-adenosyl-L-methionine
-
feed-back inhibition
S-adenosyl-L-methionine
-
feedback inhibition
Valclavam
-
oxygen analogue beta-lactam antibiotic, complete inhibition at 0.2 mM
L-methionine
-
feedback inhibition
additional information
-
overview, competitive antagonism of peptides against enzyme inhibition by valclavam; overview, inhibition of several fungi and procaryotes by valclavam
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.64
-
coenzyme A
-
recombinant protein
10
-
D-homoserine
-
recombinant protein
0.18
-
glutaryl-CoA
-
recombinant protein
0.0032
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K47A
0.027
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K46R/K47R
0.044
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant K45L
0.044
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K46A
0.049
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K47R
0.31
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant R193K
0.36
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant E237D
0.38
-
L-homoserine
-
pH 7.5, 25C, recombinant wild-type enzyme
0.58
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K46R
0.72
-
L-homoserine
-
pH 7.2, 25C, recombinant wild-type enzyme
1.07
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant C90S
1.1
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant E237A; pH 7.5, 25C, recombinant mutant R193A
1.15
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant R249A
1.51
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant R249K
1.6
-
L-homoserine
-
recombinant protein
2
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant Y238F
2.26
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant K46L
9.1
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant E246D
59.1
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant E246A
93.8
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant R193A/E246A
3.5
-
O-succinylhomoserine
-
recombinant protein
0.043
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E246D
0.05
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant Y238F/E246A
0.094
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E246A
0.094
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K47R
0.13
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K46R; pH 7.2, 25C, recombinant mutant K46R/K47R; pH 7.2, 25C, recombinant wild-type enzyme
0.14
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant C90S; pH 7.2, 25C, recombinant mutant K46A
0.15
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E237A
0.16
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K47A
0.17
-
succinyl-CoA
-
recombinant protein
0.2
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R193A/E246A
0.23
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R249A
0.24
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E237D
0.28
-
succinyl-CoA
-
pH 7.5, 25C, recombinant wild-type enzyme
0.31
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R249K
0.35
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant Y238F
0.4
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R193K
0.43
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R193A
1.78
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant K46L
2.9
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant K45L
95.5
-
L-homoserine
-
pH 7.5, 25C, recombinant mutant Y238F/E246A
additional information
-
additional information
-
steady-state kinetics and thermodynamics of wild-type and mutant enzymes, overview
-
additional information
-
additional information
-
steady-state kinetics
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
5.23
-
coenzyme A
-
recombinant protein
12
-
D-homoserine
-
recombinant protein
1.6
-
glutaryl-CoA
-
recombinant protein
0.034
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K47A
1.8
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K47R
2
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K46R/K47R
4.1
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K46A
24
-
L-homoserine
-
recombinant protein
37
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant K46R
130
-
L-homoserine
-
pH 7.2, 25C, recombinant mutant C90S; pH 7.2, 25C, recombinant wild-type enzyme
5.23
-
O-succinyl-L-homoserine
-
recombinant protein
0.34
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K47A
0.8
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E237A
1.2
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R193A/E246A
1.26
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant K46L
1.8
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K47R
2
3.7
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R249K
2
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K46R/K47R
3.3
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R193A
3.4
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R193K
4.1
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K46A
6.7
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E237D
6.9
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E246A
8.9
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant Y238F/E246A
10
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant R249A
13
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant Y238F
15
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant E246D
24
-
succinyl-CoA
-
recombinant protein
25.7
-
succinyl-CoA
-
pH 7.5, 25C, recombinant wild-type enzyme
37
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant K46R
40.1
-
succinyl-CoA
-
pH 7.5, 25C, recombinant mutant K45L
130
-
succinyl-CoA
-
pH 7.2, 25C, recombinant mutant C90S; pH 7.2, 25C, recombinant wild-type enzyme
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.00025
-
hydroxyethylvalclavam
-
dissociation constant of enzyme-substrate-inhibitor complex
-
0.00092
-
hydroxyethylvalclavam
-
-
-
0.00083
-
Valclavam
-
-
0.00089
-
Valclavam
-
dissociation constant of enzyme-substrate-inhibitor complex
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
500
-
-
wild-type histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40C, about 80% less activity as at 25C
520
-
-
mutant N267D histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40C, almost 5times higher activity at 25C
610
-
-
mutant I229T histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40C, about 4times higher activity at 25C
700
-
-
histidine-tagged enzyme, 50 mM potassium-phosphate buffer, pH 7.5, 40C, about 3.5times higher activity as at 25C
1100
-
-
mutant 333 histidine-tagged (S61T/E213V/I229T/N267D/N271K), 50 mM potassium-phosphate buffer, pH 7.5, 40C, about 50% less active as at 25C
additional information
-
-
cystathionine formation
additional information
-
-
recombinant wild-type and mutant enzymes
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.2
-
-
assay at
7.5
-
-
crude extract
7.5
-
-
assay at
additional information
-
-
broad maximum
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.4
7.5
-
25C, 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
5.4
7.5
-
25C, 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 25C without great change at about 200 micromol/min/mg protein, about 3.5times higher activity at 40C
6.2
8.5
-
pH profile of mutant K47R
6.9
8
-
crude extract, about 40% of maximal activity at pH 6.9, about 80% of maximal activity at pH 8.0
additional information
-
-
pKs of 6.6 and about 7.9
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
30
-
-
assay at
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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 40-55C but has no activity at 58C, mutants N267D and I229T show lower activity from 40-45C and similar activity profiles at 50C and beyond
40
58
-
pH 7.5, the recombinant enzyme shows a decrease in activity with temperature but still activity at 58C at about 180 microM/min/mg protein
PDB
SCOP
CATH
ORGANISM
Bacillus cereus (strain ATCC 10987)
Bacillus cereus (strain ATCC 10987)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
70000
-
-, Q72X44
gel filtration
86000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
dimer
-
2 * 35600, SDS-PAGE
dimer
-, Q72X44
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-, Q72X44
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
the transition temperature of wild-type enzyme is 47.075C, of the mutant strain 333 (S61T/E213V/I229T/N267D/N271K) 48.76C, of mutant N267D 47.36C, and of mutant I229T 46.99C
additional information
-
-
recombinant enzyme shows high transition temperature of 67.68C
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
glycerol, 30% w/v, stabilizes during purification
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
recombinant from E. coli BL21 (DE3)
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by 2 different steps of anion exchange chromatography
-
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
-
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 ACCESSION NO.
LITERATURE
expressed in Escherichia coli
-, Q72X44
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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metA gene, expression in Escherichia coli auxotrophic strains as lacZ fusion proteins
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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 44C, and mutated or wild-type gene metA transformed into Escherichia coli BL21(DE3)
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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
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ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
C142A
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site-directed mutagenesis, the mutant is inactive
C142S
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site-directed mutagenesis, the mutant is almost inactive
C142S
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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
E213V
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15-20% faster growth at 36-41C, lower growth rate at 44C (64% of control), no growth at 45C
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
H235A
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site-directed mutagenesis, the mutant is inactive
H235N
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site-directed mutagenesis, the mutant is almost inactive
H235N
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site-directed mutagenesis, the mutant is inactive
I229T
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normal growth at 37C, better growth at 44C compared to control, 48% faster growth at 44C, 18% faster growth at 43C, no growth at 45C, 1.4times greater cell density with 30 mM acetic acid at 30C than control, heating at 45C 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 30C 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
K156L
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site-directed mutagenesis, the mutant is inactive
K45A/K46A
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no residual enzyme activity, strain is auxotroph for L-methionine
K45A/K46A
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site-directed mutagenesis, the mutant is inactive
K45A/K46A
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site-directed mutagenesis, the mutant is nearly inactive
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
N267D
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normal growth at 37C, better growth at 44C compared to control, 31% faster growth at 44C, 10% faster growth at 43C, no growth at 45C, with 30 mM acetic acid at 30C slower growth than other thermostable strains and only 16% higher cell density than control, heating at 45C 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 30C 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 36-41C, lower growth rate at 44C (22% of control), no growth at 45C
P298L
-
point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A5
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 36-41C (15-20% faster), similar growth as wild-type at elevated temperatures, no growth at 45C
S61T/E213V/I229T/N267D/N271K
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introduction of random mutagenesis by error-prone PCR, fast growing strain at 44C, 5-12% faster growing than control strain at a temperature range from 30-42C, at 43C 30% faster growth, at 44C 64% faster growth, no growth at 45C, 1.4fold slower growth in methionine deficient medium at 43 and 44C compared to 1.6 and 2fold slowing in the wild-type, 1.4times greater cell density with 30 mM acetic acid at 30C than control, heating at 45C 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 30C shows 1.5-3times higher levels of soluble enzyme than the wild-type strain and 9times higher insoluble enzyme levels
Y238F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Y238F/E246A
-
site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency with L-homoserine, but increased with succinyl-CoA compared to the wild-type enzyme
P298L
Escherichia coli W3110
-
point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A5
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K47R
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site-directed mutagenesis, the mutant shows 90% reduced activity compared to the wild-type enzyme
additional information
-
construction of IS-insertion mutant metA7, with 298Pro-Tyr-Asp-Leu-Arg-His-Met-Asn-Pro-Thr-Leu-Asp-stop to 298Arg-Leu-Ala-Pro-stop, obtained from norleucine-resistant strain, and IS insertion mutant metA8, with 298Pro-Tyr-Asp-Leu-Arg-His-Met-Asn-Pro-Thr-Leu-Asp-stop to 298Arg-Leu-Ala-Pro-stop, also obtained from norleucine-resistant strain, overview, introduction of deletion mutations of metJ and thrBC into the W3110 strain has a significant effect on the amount of Met excreted into the medium, overview
additional information
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wild-type Escherichia coli enzyme, normal growth at 37C, less growth than the mutants at 44C no growth at 45C, heating at 45C 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 30C shows 1.5-3times lower levels of soluble enzyme than the mutants and 15times higher insoluble enzyme levels
I296S
Escherichia coli W3110
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A4
-
additional information
Escherichia coli W3110
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construction of IS-insertion mutant metA7, with 298Pro-Tyr-Asp-Leu-Arg-His-Met-Asn-Pro-Thr-Leu-Asp-stop to 298Arg-Leu-Ala-Pro-stop, obtained from norleucine-resistant strain, and IS insertion mutant metA8, with 298Pro-Tyr-Asp-Leu-Arg-His-Met-Asn-Pro-Thr-Leu-Asp-stop to 298Arg-Leu-Ala-Pro-stop, also obtained from norleucine-resistant strain, overview, introduction of deletion mutations of metJ and thrBC into the W3110 strain has a significant effect on the amount of Met excreted into the medium, overview
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R27C
Escherichia coli W3110
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point mutation in gene metA leading to a deregulated methionine biosynthesis in the mutant strain A9
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additional information
-
the recombinant Geobacillus kaustophilus enzyme stimulates Escherichia coli growth at 44C (20% faster than control) but inhibits growth by 20% at 37C compared to the wild-type, between 30-42C growth is inhibited by about 10%, at 43C growth is equal to the Escherichia coli wild-type enzyme
additional information
Geobacillus kaustophilus KCTC 3397
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the recombinant Geobacillus kaustophilus enzyme stimulates Escherichia coli growth at 44C (20% faster than control) but inhibits growth by 20% at 37C compared to the wild-type, between 30-42C growth is inhibited by about 10%, at 43C growth is equal to the Escherichia coli wild-type enzyme
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APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
biotechnology
-
enzyme methionine biosynthesis deregulation mutant strains are useful for construction/production of L-methionine excreting strains
biotechnology
Escherichia coli W3110
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enzyme methionine biosynthesis deregulation mutant strains are useful for construction/production of L-methionine excreting strains
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