Information on EC 4.3.1.19 - threonine ammonia-lyase

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

EC NUMBER
COMMENTARY hide
4.3.1.19
-
RECOMMENDED NAME
GeneOntology No.
threonine ammonia-lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-aminobut-2-enoate = 2-iminobutanoate
show the reaction diagram
(1b), spontaneous
-
-
-
2-iminobutanoate + H2O = 2-oxobutanoate + NH3
show the reaction diagram
(1c), spontaneous
-
-
-
L-threonine = 2-aminobut-2-enoate + H2O
show the reaction diagram
(1a)
-
-
-
L-threonine = 2-oxobutanoate + NH3
show the reaction diagram
(overall reaction); that from P. putida is not. The enzyme from a number of sources also acts on L-serine, cf. EC 4.3.1.17, L-serine ammonia-lyase. The reaction catalysed probably involves initial elimination of water, hence the enzyme's original classification as EC 4.2.1.16, threonine dehydratase, followed by isomerization and hydrolysis of the product with C-N bond breakage; The enzyme from many sources is a pyridoxal-phosphate protein
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Deamination
-
-
-
-
elimination
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
Glycine, serine and threonine metabolism
-
-
L-threonine degradation V
-
-
Metabolic pathways
-
-
serine metabolism
-
-
threonine metabolism
-
-
Valine, leucine and isoleucine biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
L-threonine ammonia-lyase (2-oxobutanoate-forming)
Most enzymes that catalyse this reaction are pyridoxal-phosphate-dependent, although some enzymes contain an iron-sulfur cluster instead. The reaction catalysed by both types of enzymes involves the initial elimination of water to form an enamine intermediate (hence the enzyme's original classification as EC 4.2.1.16, threonine dehydratase), followed by tautomerization to an imine form and hydrolysis of the C-N bond [3,5]. The latter reaction, which can occur spontaneously, is also be catalysed by EC 3.5.99.10, 2-iminobutanoate/2-iminopropanoate deaminase [5]. The enzymes from a number of sources also act on L-serine, cf. EC 4.3.1.17, L-serine ammonia-lyase.
CAS REGISTRY NUMBER
COMMENTARY hide
9024-34-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Anacystis marina
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain Ok-79-fl
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain F5, N.C.I.B. 11102
-
-
Manually annotated by BRENDA team
strain F5, N.C.I.B. 11102
-
-
Manually annotated by BRENDA team
Japanese quail, male
-
-
Manually annotated by BRENDA team
Cyclotella nana
-
-
-
Manually annotated by BRENDA team
strain K 12
-
-
Manually annotated by BRENDA team
strain SM-ZK, dehydratase I and dehydratase II
-
-
Manually annotated by BRENDA team
strain SM-ZK, dehydratase I and dehydratase II
-
-
Manually annotated by BRENDA team
strain DL-1, ATCC 51573, gene GSU0486 or tcdB
UniProt
Manually annotated by BRENDA team
strain DL-1, ATCC 51573, gene GSU0486 or tcdB
UniProt
Manually annotated by BRENDA team
Hansenula henricii
-
-
-
Manually annotated by BRENDA team
strain AKU 0084,biosynthetic threonine deaminase
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain PAC1
-
-
Manually annotated by BRENDA team
strain PAC1
-
-
Manually annotated by BRENDA team
strain Lz4W, gene ilvA
-
-
Manually annotated by BRENDA team
strain Lz4W, gene ilvA
-
-
Manually annotated by BRENDA team
biosynthetic threonine deaminase
-
-
Manually annotated by BRENDA team
Rhodosporidium toruloides
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
i.e. Agmenellum quadruplicatum
-
-
Manually annotated by BRENDA team
Tetraselmis maculata
-
-
-
Manually annotated by BRENDA team
X-1
-
-
Manually annotated by BRENDA team
X-1
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
-
SP0454 contributes to virulence and colonization
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
beta-chloro-L-Ala
?
show the reaction diagram
DL-allo-cystathionine
?
show the reaction diagram
-
at 20% of the activity with L-Thr
-
-
-
L-allothreonine
?
show the reaction diagram
L-Cys
?
show the reaction diagram
L-homoserine
2-oxobutanoate + NH3 + H2O
show the reaction diagram
-
-
-
-
?
L-Ser
pyruvate + NH3
show the reaction diagram
-
-
-
-
ir
L-serine
pyruvate + NH3
show the reaction diagram
L-Thr
2-oxobutanoate + NH3
show the reaction diagram
-
-
-
-
?
L-Thr
?
show the reaction diagram
L-threonine
2-oxobutanoate + NH3
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
L-serine
pyruvate + NH3
show the reaction diagram
-
-
-
?
L-Thr
?
show the reaction diagram
L-threonine
2-oxobutanoate + NH3
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyridoxal 5'-phosphate
pyridoxamine 5'-phosphate
-
reactivates after dissociation of the coenzyme
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Na+
-
1.7fold increase in activity
Rb+
-
can partially replace K+ in activation
additional information
-
no requirement for divalent cations
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,2'-dithiodipyridine
2-oxobutanoate
2-oxoglutarate
-
-
bicarbonate
-
-
Biliverdin
-
-
Carbamate
-
-
cysteamine
-
-
dithiothreitol
formaldehyde
-
-
glyoxylate
homoserine
-
-
hydrazine
hydroxylamine
iodoacetamide
iodoacetate
-
-
isoleucine
Isoniazid
L-Cys
-
competitive
L-isoleucine
methoxylamine
Na-chenodeoxycholate
-
-
Na-cholate
-
-
Na-cholatemethyl ester
-
-
Na-deoxycholate
-
-
Na-lithocholate
-
-
NSD-1055
p-Chloromercuriphenyl sulfonate
phenylhydrazine
phosphoenolpyruvate
-
-
pyruvate
Semicarbazide
Urea
-
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
isoleucine
-
activation below 0.01 mM, strong inhibition above, 50% inhibition at 0.064 mM
L-valine
-
-
NH4+
-
2.1fold increase in activity
phosphate
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.25 - 380
L-Ser
3
L-serine
processed TD2
0.25 - 129
L-Thr
2.3 - 123
L-threonine
additional information
additional information
-
Monod-Wyman-Changeux symmetrical model analysis of steady-state kinetics for the wild-type and four mutant enzymes
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3 - 243
L-Ser
5.2 - 287
L-Thr
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2
L-Ser
Entamoeba histolytica
-
pH 9.0, 37C
262
0.7
L-Thr
Entamoeba histolytica
-
pH 9.0, 37C
780
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.06 - 0.14
Ile
1.1 - 2.2
L-Cys
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.009 - 6.99
Ile
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.52
-
-
400
-
recombinant threonine deaminase
1000
-
pH 8, 22C
1470
-
pH 8, 22C
7400
-
pH 8, 22C
9900
-
pH 8, 22C
23000
-
pH 8, 22C
106000
-
pH 8, 22C
171000
-
pH 8, 22C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4 - 9.2
-
in presence of AMP
8 - 9
-
-
8 - 9
-
Tris/HCl buffer and diethanolamine buffer
8
-
assay at
8 - 9.5
-
in presence of 1 mM AMP
8.7
Tetraselmis maculata
-
-
9 - 9.8
-
in absence of AMP
9.2 - 9.6
9.4 - 9.6
-
-
9.5 - 11
-
dehydratase I
10
-
with L-Thr and L-Ser as substrate
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8
-
65% of maximal activity at pH 7 and at pH 8
7.5 - 9.5
8.1 - 9.6
-
50% of maximal activity at pH 8.1 and 9.6
8.5 - 11
-
pH 8.5: about 30% of maximal activity, pH 11.0: about 70% of maximal activity, with L-Thr as substrate
additional information
the enzyme is highly active in an alkaline pH range, little or no activity is observed at pH values below 6.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
assay at
35
-
in absence of phosphate
44
-
in presence of 250 mM phosphate
85 - 90
-
-
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45 - 80
-
45C: about 25% of maximal activity, 80C: about 35% of maximal activity
60 - 98
-
60C: about 30% of maximal activity, 98C: about 15% of maximal activity
additional information
active over a wide range of temperatures, optimal enzyme activity is observed at 58C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.7
-
calculated
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
primary culture
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
molecular mass of processed TD2 determined by SDS-PAGE
45000
-
determined by gel-filtration in the absence of CMP and AMP
55000
determined by Western blot analysis, mature TD2
66180
predicted molecular mass of SlTD1
100000 - 115000
-
gel filtration
100000
-
determined by gel-filtration in the presence of CMP
106000
-
gel filtration
118000
-
gel filtration
120000
140000
-
native PAGE, in the presence of the allosteric effector isoleucine
147000
-
sedimentation equilibrium ultracentrifugation
160000
-
gel filtration
190000
200000
201000
-
equilibrium sedimentation
203800
-
sedimentation equilibrium experiments
210000
214000
-
meniscus depletion equilibrium sedimentation, analytical ultracentrifugation
228000
-
calculation from sedimentation and diffusion data
230000
-
gel filtration
250000
-
dehydratase II, gel filtration
268000
-
native PAGE, in the absence of allosteric effector
370000
-
Ile-sensitive enzyme form, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 45000, calculated
tetramer
additional information
-
the short C-terminal regulatory domain is composed of only one ACT-like subdomain
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure at 2.8 A resolution
-
data for two crystal forms are collected to resolutions of 2.2 and 1.7 A, crystals obtainted in the presence of CMP diffract to a resolution of 3-3.5 A, in the presence of AMP poorly
-
two data sets of resolutions 2.2 A, crystal form I, and 1.7 A, crystal form II, are collected
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
10 h, 81% loss of activity in presence of 0.05 mM Ile, stable in presence of 1 mM Ile
27
-
10 h, 27% loss of activity in presence of 0.05 mM Ile, stable in presence of 1 mM Ile
37
-
10 min, 16% loss of activity of dehydratase I and 36% loss of activity of dehydratase II, loss of activity of dehydratase I is prevented by 1 mM Ile, but that of dehydratase II is not
45
-
15 min, complete loss of activity in absence of phosphate, 94% loss of activity in presence of 50 mM phosphate, 49% loss of activity in presence of 250 mM phosphate
70
-
1 h, 15% loss of activity
90
-
rapid inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dithiothreitol, allothreonine and pyridoxal phosphate are all required to maintain a stable form of threonine dehydratase
-
L-Ile protects the enzyme against inactivation at low temperatures
-
loss of activity of dehydratase I at 37 C is prevented by 1 mM Ile, but that of dehydratase II is not
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 0.05 M potassium phosphate, pH 7.2, enzyme concentration 0.06 mg/ml, half-life: 4 weeks
-
-20C, 50% loss of activity after 3 weeks
-
-20C, no loss of activity after several weeks
-
0-4C, stable for at least 2 months
-
0C, rapid loss of activity unless maintained in presence of Ile and potassium phosphate
-
4C, little loss of activity after 50 days
-
4C, loss of activity after storage of more than one day
-
4C, pH 7.2 or 9.0, 24 h, complete inactivation after 7 days, stable in presence of egg albumin
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant strain DU-21 with an activator site-deficient enzyme form
-
partial
recombinant threonine deaminase
-
using a nickel-nitrilotriacetic acid affinity column
-
using a Sephadex G-25, a DEAE-cellulose DE52, a HiLoad26/60 Superdex 200 and a HiPrep 16/10 column
using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a modified SlTD2 cDNA is cloned into the pET30a+ vector producing a truncated form of SlTD2 lacking the 51 amino acids corresponding to the N-terminal chloroplast-targeting sequence
expressed in Escherichia coli as a His-tagged fusion protein
expression in Brevibacterium flavum
-
expression in Brevibacterium flavum; expression in Corynebacterium glutamicum
-
expression in Escherichia coli
-
expression of tdcB in Corynebacterium glutamicum ATCC 21799
-
gene tcdB, phylogenetic analysis of threonine ammonia-lyases
ilvA expression in Nicotiana tabaccum is effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with L-O-methylthreonine as the selction agent
-
into the pRSETC vector for expression in Escherichia coli BL21DE3 pLysS cells
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
about 500 times higher transcript levels in flowers than in leaves or roots; induced transcription in response to osmotic stress
-
induced transcription in response to osmotic stress
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E520A
-
omr1-7 allele, tolerates high concentrations of L-O-methylthreonine
H542L
-
omr1-8 allele, tolerates high concentrations of L-O-methylthreonine
R499C
-
omr1-5 allele, tolerates high concentrations of L-O-methylthreonine
R499C/R544H
-
omr1-1 allele, tolerates high concentrations of L-O-methylthreonine
Y449L
-
concentration of isoleucine needed to reach 50% inhibition increases by a factor 45, two different effector-binding sites are constituted in part by Y449 and Y543
Y543L
-
concentration of isoleucine needed to reach 50% inhibition increases by a factor 38, two different effector-binding sites are constituted in part by Y449 and Y543
G350A
-
site-directed mutagenesis, the affinity for both allosteric effectors is lower compared to the wild-type, valine binds exclusively to the R state, the mutation causes a shift in the equilibrium between the T and R conformational states of the protein toward the T state with L being higher than that of the wild-type enzyme
L352A
-
site-directed mutagenesis, the affinity for both allosteric effectors is lower compared to the wild-type, valine binds exclusively to the R state, the mutation causes a shift in the equilibrium between the T and R conformational states of the protein toward the T state with L being 6.5fold higher than that of the wild-type enzyme
N363A
-
site-directed mutagenesis, the mutant acts similar to the wild-type
Q347A
-
site-directed mutagenesis, mutant Q347A is very similar to the wild-type enzyme in most of its characteristics, except for a 1.5fold increase in L and a 5fold increase in KTIle
T367A
-
site-directed mutagenesis, the T367A mutation causes a decrease in the affinity of bsTD for both allosteric effectors and an increase in substrate affinity compared to the wild-type enzyme
Y371L
-
site-directed mutagenesis, the apparent affinities for both of the allosteric effectors are very low and the apparent dissociation constant for isoleucine from the T state is 50fold higher compared to the wild-type
Val323Ala
-
feedback inhibition by L-Ile is entirely abolished, so that the enzyme is always present in a relaxed high-activity state
E347F
-
mutation decreases the K0.5 values of Thr without significant change of the n(H) value compared to wild-type. Mutant is strongly feedback-resistant to Ile compared to the wild-type enzyme. IC50 (Ile) is increased compared to wild-type
E442A
-
mutation increases the K0.5 value of Thr and n(H) value, comparing to those of the wild-type enzyme. IC50 (Ile) is decreased compared to wild-type
F352A
-
mutation decreases the K0.5 values of Thr without significant change of the n(H) value compared to wild-type. Mutant is strongly feedback-resistant to Ile compared to the wild-type enzyme. IC50 (Ile) is increased compared to wild-type
F352A/I460F
-
double mutant is more resistant to Ile inhibition than any single site mutant. Double mutations retains more than 85% activity even at 10 mM Ile
F352A/R362F
-
mutant shows both higher activity and stronger resistance to Ile inhibition compared to wild-type mice. Overexpression of mutant in Escherichia coli JW3591 significantly increases the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type
G350E
-
mutation increases the K0.5 value of Thr and n(H) value, comparing to those of the wild-type enzyme. IC50 (Ile) is decreased compared to wild-type
G445E
-
mutation G445E increased the K0.5 value of Thr without change of n(H) value compared to wild-type. IC50 (Ile) is slightly increased compared to wild-type
I460F
-
mutation decreases the K0.5 values of Thr without significant change of the n(H) value compared to wild-type. Mutant is strongly feedback-resistant to Ile compared to the wild-type enzyme. IC50 (Ile) is increased compared to wild-type
I460F/R362F
-
double mutant is more resistant to Ile inhibition than any single site mutant. Double mutations retains more than 85% activity even at 10 mM Ile
R362F
-
mutation decreases the K0.5 values of Thr without significant change of the n(H) value compared to wild-type. Mutant is strongly feedback-resistant to Ile compared to the wild-type enzyme. IC50 (Ile) is increased compared to wild-type
Y369L
-
mutant shows an 91fold increased IC50 (Ile) value compared to wild-type
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
-
in contrast to the wild-type, all four transgenic TD lines are able to tolerate high concentrations of L-O-methylthreonine. This illustrates the potential use of these mutant omr genes as dominant selectable markers in plant transformation
synthesis
-
production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from a single unrelated carbon source via threonine biosynthesis in Escherichia coli, by overexpression of threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, removal of the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in Escherichia coli, and knock-out of the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Construction of a series of strains and mutants leads to production of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer is 17.5 mol%
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