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 hide
2.3.1.46
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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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-
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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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methionine metabolism
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Sulfur 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
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 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
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
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-
-
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r
succinyl-CoA + D-homoserine
CoA + O-succinyl-D-homoserine
show the reaction diagram
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-
-
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r
succinyl-CoA + L-homoserine
CoA + O-succinyl-L-homoserine
show the reaction diagram
additional information
?
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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
?
-
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
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
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00025 - 0.00092
hydroxyethylvalclavam
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, 40C, about 80% less activity as at 25C
520
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mutant N267D histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40C, almost 5times higher activity at 25C
610
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mutant I229T histidine-tagged, 50 mM potassium-phosphate buffer, pH 7.5, 40C, about 4times higher activity at 25C
700
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histidine-tagged enzyme, 50 mM potassium-phosphate buffer, pH 7.5, 40C, about 3.5times higher activity as at 25C
1100
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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
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
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
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
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
glycerol, 30% w/v, stabilizes during purification
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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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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 44C, and mutated or wild-type gene metA transformed into Escherichia coli BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
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
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
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
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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
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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
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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