Information on EC 4.1.2.48 - low-specificity L-threonine aldolase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
4.1.2.48
-
RECOMMENDED NAME
GeneOntology No.
low-specificity L-threonine aldolase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-allo-threonine = glycine + acetaldehyde
show the reaction diagram
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-
-
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L-threonine = glycine + acetaldehyde
show the reaction diagram
(1)
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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Biosynthesis of secondary metabolites
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glycine biosynthesis IV
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Glycine, serine and threonine metabolism
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L-threonine degradation IV
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
L-threonine/L-allo-threonine acetaldehyde-lyase (glycine-forming)
Requires pyridoxal phosphate. The low-specificity L-threonine aldolase can act on both L-threonine and L-allo-threonine [1,2]. The enzyme from Escherichia coli can also act on L-threo-phenylserine and L-erythro-phenylserine [4]. The enzyme can also catalyse the aldol condensation of glycolaldehyde and glycine to form 4-hydroxy-L-threonine, an intermediate of pyridoxal phosphate biosynthesis [3]. Different from EC 4.1.2.5, L-threonine aldolase, and EC 4.1.2.49, L-allo-threonine aldolase.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain SGY269
SwissProt
Manually annotated by BRENDA team
strain SGY269
SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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E7U392
UniProt
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
strain A3(2)
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-
Manually annotated by BRENDA team
strain A3(2)
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
knockout of the ltaE gene of wild-type Escherichia coli does not affect the cellular growth rate, while disruption of the ltaE gene of Escherichia coli GS245, whose serine hydroxymethyltransferase gene is knocked out, causes a significant decrease in the cellular growth rate, suggesting that the threonine aldolase is not a major source of cellular glycine in wild-type Escherichia coli but catalyzes an alternative pathway for cellular glycine when serine hydroxymethyltransferase is inert
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3,4-dihydroxybenzaldehyde + glycine
(2S,3R,4R)-2-amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic acid
show the reaction diagram
3,4-dihydroxybenzaldehyde + glycine
(2S,3S,4R)-2-amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic acid
show the reaction diagram
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conversion: 40%, glycine concentration: 70 mM, reaction temperature: 4C, yield: 30%, L-erythro/L-threo: 16:84
-
-
?
benzyloxyacetaldehyde + glycine
(2S,3R)-2-amino-4-(benzyloxy)-3-hydroxybutanoic acid
show the reaction diagram
benzyloxyacetaldehyde + glycine
(2S,3S)-2-amino-4-(benzyloxy)-3-hydroxybutanoic acid
show the reaction diagram
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conversion: 45%, glycine concentration: 140 mM, reaction temperature: 25C, yield: 30%, L-erythro/L-threo: 40:60
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-
?
DL-erythro-phenylserine
glycine + benzaldehyde
show the reaction diagram
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-
-
?
DL-threo-(3-methylsulfonylphenyl)serine
glycine + 3-methylsulfonylbenzaldehyde
show the reaction diagram
121% of the activity with L-threonine
-
-
?
DL-threo-(3-nitrophenyl)serine
glycine + 3-nitrobenzaldehyde
show the reaction diagram
143% of the activity with L-threonine
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-
?
DL-threo-phenylserine
glycine + benzaldehyde
show the reaction diagram
glycine + 3,4-dihydroxybenzaldehyde
L-threo-3,4-dihydroxyphenylserine
show the reaction diagram
glycine + 3,4-dihydroxybenzaldehyde
L-threo-3,4-dihydroxyphenylserine + L-erythro-3,4-dihydroxyphenylserine
show the reaction diagram
glycine + glycolaldehyde
L-4-hydroxythreonine
show the reaction diagram
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low-specificity L-threonine aldolase is involved in a serendipitous pathway that converts 3-phosphohydroxypyruvate, an intermediate in the serine biosynthesis pathway, to L-4-phosphohydroxythreonine, an intermediate in the pyridoxal-5'-phosphate synthesis pathway in a strain of Escherichia coli that lacks 4-phosphoerythronate dehydrogenase
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r
L-4-hydroxythreonine
glycine + glycolaldehyde
show the reaction diagram
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cleavage of L-4-hydroxythreonine is as efficient as cleavage of L-allo-threonine
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r
L-allo-threonine
glycine + acetaldehyde
show the reaction diagram
L-erythro-phenylserine
glycine + benzaldehyde
show the reaction diagram
L-phenylserine
?
show the reaction diagram
L-serine
glycine + formaldehyde
show the reaction diagram
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-
-
-
?
L-Thr
Gly + acetaldehyde
show the reaction diagram
L-threo-3,4-dihydroxyphenylserine
glycine + 3,4-dihydroxybenzaldehyde
show the reaction diagram
L-threo-beta-3,4-dihydroxyphenylserine
glycine + 3,4-dihydroxybenzaldehyde
show the reaction diagram
L-threo-beta-3,4-methylenedioxyphenylserine
?
show the reaction diagram
L-threo-phenylserine
glycine + benzaldehyde
show the reaction diagram
-
-
-
?
L-threonine
glycine + acetaldehyde
show the reaction diagram
N-(S)-benzyloxycarbonyl-alaninal + glycine
(2S,3R,4S)-2-amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic acid
show the reaction diagram
N-(S)-benzyloxycarbonyl-alaninal + glycine
(2S,3S,4S)-2-amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic acid
show the reaction diagram
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conversion: 54%, glycine concentration: 140 mM, reaction temperature: 25C, yield: 27%, L-erythro/L-threo: 18:82
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-
?
N-benzyloxycarbonyl-3-aminopropanal + glycine
(2S,3R)-2-amino-5-(benzyloxycarbonylamino)-3-hydroxypentanoic acid
show the reaction diagram
N-benzyloxycarbonyl-3-aminopropanal + glycine
(2S,3S)-2-amino-5-(benzyloxycarbonylamino)-3-hydroxypentanoic acid
show the reaction diagram
N-benzyloxycarbonyl-glycinal + glycine
(2S,3R)-2-amino-4-(benzyloxycarbonylamino)-3-hydroxybutanoic acid
show the reaction diagram
N-benzyloxycarbonyl-glycinal + glycine
(2S,3S)-2-amino-4-(benzyloxycarbonylamino)-3-hydroxybutanoic acid
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
glycine + glycolaldehyde
L-4-hydroxythreonine
show the reaction diagram
-
low-specificity L-threonine aldolase is involved in a serendipitous pathway that converts 3-phosphohydroxypyruvate, an intermediate in the serine biosynthesis pathway, to L-4-phosphohydroxythreonine, an intermediate in the pyridoxal-5'-phosphate synthesis pathway in a strain of Escherichia coli that lacks 4-phosphoerythronate dehydrogenase
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-
r
L-Thr
Gly + acetaldehyde
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyridoxal 5'-phosphate
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
-
K+ or NH4+ required for maximal activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ba2+
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inhibited activation by K+ or NH4+
Ca2+
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inhibited activation by K+ or NH4+
Mg2+
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inhibited activation by K+ or NH4+
Na+
-
inhibited activation by K+ or NH4+
tetrahydrofolate
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inhibits interconversion of L-serine and glycine
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NH4+
-
K+ or NH4+ required for maximal activity
sodium sulfite
addition of sodium sulfite stimulates the production of L-threo-3,4-dihydroxyphenylserine without affecting the diastereoselectivity ratio, especially at 50 mM
Triton X-100
highest conversion yield at a 0.75% without affecting the diastereoselectivity ratio
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.24
DL-erythro-phenylserine
pH 8.0, 30C
23.6
DL-threo-(3-methylsulfonylphenyl)serine
pH 7.0, 37C
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21.5
DL-threo-(3-nitrophenyl)serine
pH 7.0, 37C
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0.12 - 19.2
DL-threo-phenylserine
0.027
L-4-hydroxythreonine
-
pH 8.0, 25C
0.052 - 31
L-allo-threonine
10.2
L-erythro-phenylserine
pH 8.0, 30C
0.000002 - 0.0000026
L-phenylserine
0.00016 - 0.00018
L-Thr
0.0002 - 0.00023
L-threo-3,4-dihydroxyphenylserine
8.3
L-threo-beta-3,4-dihydroxyphenylserine
pH 8.0, 30C
7.4
L-threo-beta-3,4-methylenedioxyphenylserine
pH 8.0, 30C
7.3
L-threo-phenylserine
pH 8.0, 30C
0.4 - 63
L-threonine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.44
L-4-hydroxythreonine
Escherichia coli
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pH 8.0, 25C
0.06 - 41
L-allo-threonine
0.000107 - 0.000113
L-phenylserine
0.0178 - 0.0195
L-Thr
0.00022 - 0.00023
L-threo-3,4-dihydroxyphenylserine
0.02 - 43
L-threonine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.3
L-4-hydroxythreonine
Escherichia coli
-
pH 8.0, 25C
28479
0.01 - 45.08
L-allo-threonine
0.0016 - 3.53
L-threonine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.7
pH 8.0, 30C
8.5
cell-free extract
41
pH 8.0, 30C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8
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recombinant enzyme
8.5 - 9
10
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substrate: L-allo-threonine, Tris-chloride buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
-
recombinant enzyme
55
aldehyde formation from L-allo-threonine
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37000
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subunit, SDS-PAGE
41810
calculated from amino acid sequence
140000
gel filtration
145000
gel filtration
150000
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gel filtration
170000
gel filtration
277000
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ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at 2.2 A resolution, in the unliganded form and cocrystallized with L-serine and L-threonine. No active site catalytic residue is revealed, and a structural water molecule is assumed to act as the catalytic base in the retro-aldol cleavage reaction. The very large active site opening suggests that much larger molecules than L-threonine isomers may be easily accommodated
E7U392
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9
30C, 30 min, stable
441433
6 - 9.5
30C, 30 min, stable
5206
6.5 - 10
30 min, stable
5205
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
40C, 15 min, 50% loss of activity
50
15 min, enzyme retains only 25% of activity
63
-
the half-life of the wild type L-TA at 63C is 1.3 min
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Hiprep 16/10 DEAE FF column chromatography, Super Sepharose column chromatography, and HiLoad 16/10 Phenyl Sepharose HP column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into pUCl18, and expressed in Escherichia coli
expressed in Escherichia coli strain JM109
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expression in Escherichia coli
overexpression in Escherichia coli
recombinantly expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D176E
500fold decrease in catalytic efficiency
D95C
less than 10% of catalytic efficiency of wild-type
D95H/E96G
less than 10% of catalytic efficiency of wild-type
D95L
less than 10% of catalytic efficiency of wild-type
D95M
less than 5% of catalytic efficiency of wild-type
D95N/E96S
less than 5% of catalytic efficiency of wild-type
D95W
less than 2% of catalytic efficiency of wild-type
D95Y
less than 5% of catalytic efficiency of wild-type
D95Y/E96T
less than 10% of catalytic efficiency of wild-type
D176E
-
500fold decrease in catalytic efficiency
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D95C
-
less than 10% of catalytic efficiency of wild-type
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D95L
-
less than 10% of catalytic efficiency of wild-type
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D95M
-
less than 5% of catalytic efficiency of wild-type
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D95Y
-
less than 5% of catalytic efficiency of wild-type
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F87A
E7U392
no change in the ration of cleavage of L-threonine to L-allo-threonine
F87D
E7U392
mutation doubles the preference of the enzyme for L-allo-threonine
H126F
E7U392
300% of wild-type activity,reduced preference for the erythro-substrate
H126N
E7U392
60% of wild-type activity
H83F
E7U392
less than 1% of wild-type activity, reduced preference for the erythro-substrate
H83F/H126F
E7U392
able to catalyze the cleavage of both L-threonine and L-allo-threonine at a measurable rate, neither of the histidines acts as a catalytic base in the retro-aldol cleavage mechanism
H83N
E7U392
less than 10% of wild-type activity
K222A
E7U392
slight decrease in kcat and slight increase in Km values for both L-threonine and L-allo-threonine
F87A
-
no change in the ration of cleavage of L-threonine to L-allo-threonine
-
F87D
-
mutation doubles the preference of the enzyme for L-allo-threonine
-
H126F
-
300% of wild-type activity,reduced preference for the erythro-substrate
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H126N
-
60% of wild-type activity
-
K222A
-
slight decrease in kcat and slight increase in Km values for both L-threonine and L-allo-threonine
-
K207A
the mutant enzyme shows no detectable enzyme activity. The mutant enzyme show the disappearance of the absorption maximum at 420 nm, indicating that the Schiff base linkage between the epsilon-amino group of the active-site lysine residue and the pyridoxal 5'-phosphate cofactor aldehyde group of the wild type is not present in the mutant enzyme
K207R
the mutant enzyme shows a specific activity of about 1000 times lower than that of the wild-type enzyme. The mutant enzyme show the disappearance of the absorption maximum at 420 nm, indicating that the Schiff base linkage between the epsilon-amino group of the active-site lysine residue and the pyridoxal 5'-phosphate cofactor aldehyde group of the wild type is not present in the mutant enzyme
K207A
-
the mutant enzyme shows no detectable enzyme activity. The mutant enzyme show the disappearance of the absorption maximum at 420 nm, indicating that the Schiff base linkage between the epsilon-amino group of the active-site lysine residue and the pyridoxal 5'-phosphate cofactor aldehyde group of the wild type is not present in the mutant enzyme
-
K207R
-
the mutant enzyme shows a specific activity of about 1000 times lower than that of the wild-type enzyme. The mutant enzyme show the disappearance of the absorption maximum at 420 nm, indicating that the Schiff base linkage between the epsilon-amino group of the active-site lysine residue and the pyridoxal 5'-phosphate cofactor aldehyde group of the wild type is not present in the mutant enzyme
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A169T
-
stability-enhanced mutant, half life at 63C is 3.7 min
D104N
-
stability-enhanced mutant, half life at 63C is 5.8 min
F18I
-
stability-enhanced mutant, half life at 63C is 5.0 min, the specific activity is decreased by 45% compared to the wild type enzyme
H177Y
-
stability-enhanced mutant, half life at 63C is 14.6 min
R241C/A287V
-
the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
V86I/R241C/Y306C
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the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
Y34C
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the mutation dramatically increases the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
Y39C/Y306C
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the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
Y39C/Y306C/A48T
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the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
Y39C/Y306C/R316C
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the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
A169T
-
stability-enhanced mutant, half life at 63C is 3.7 min
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D104N
-
stability-enhanced mutant, half life at 63C is 5.8 min
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F18I
-
stability-enhanced mutant, half life at 63C is 5.0 min, the specific activity is decreased by 45% compared to the wild type enzyme
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H177Y
-
stability-enhanced mutant, half life at 63C is 14.6 min
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R241C/A287V
-
the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
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V86I/R241C/Y306C
-
the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
-
Y34C
-
the mutation dramatically increases the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
-
Y39C/Y306C
-
the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
-
Y39C/Y306C/A48T
-
the mutations dramatically increase the diastereoselectivity of the reverse aldol condensation activity for L-threo-3,4-dihydroxyphenylserine
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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a continuous bioconversion system for L-threo-3,4-dihydroxyphenylserine production is developed that uses whole-cell biocatalyst of recombinant Escherichia coli expressing L-TA genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates are observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g Escherichia coli cells/l, pH 6.5 and 10C. In the optimized condition, overall productivity is 8 g/l
synthesis
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