Information on EC 4.3.1.18 - D-Serine ammonia-lyase

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

EC NUMBER
COMMENTARY hide
4.3.1.18
-
RECOMMENDED NAME
GeneOntology No.
D-Serine ammonia-lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-Serine = pyruvate + NH3
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Glycine, serine and threonine metabolism
-
-
serine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
D-serine ammonia-lyase (pyruvate-forming)
A pyridoxal-phosphate protein. The enzyme cleaves a carbon-oxygen bond, releasing a water molecule (hence the enzyme's original classification as EC 4.2.1.14, D-serine dehydratase) and an unstable enamine product that tautomerizes to an imine form, which undergoes a hydrolytic deamination to form pyruvate and ammonia. The latter reaction, which can occur spontaneously, can also be catalysed by EC 3.5.99.10, 2-iminobutanoate/2-iminopropanoate deaminase. Also acts, slowly, on D-threonine.
CAS REGISTRY NUMBER
COMMENTARY hide
9015-88-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain A-21
-
-
Manually annotated by BRENDA team
strain A-21
-
-
Manually annotated by BRENDA team
strain CFT073, recombinant enzyme
-
-
Manually annotated by BRENDA team
K12
SwissProt
Manually annotated by BRENDA team
Sprague-Dawley male rat
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
a dsdA mutant is attenuated in virulence
physiological function
DsdA contributes to virulence within the urinary tract of mice
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-D-alanine
3-chloro-D-alanine
show the reaction diagram
-
activity of approximately 2%
-
-
?
beta-chloro-D-alanine
?
show the reaction diagram
-
-
-
?
D-allo-Thr
2-oxobutanoate + NH3
show the reaction diagram
-
-
-
-
?
D-allo-threonine
?
show the reaction diagram
-
-
-
?
D-Allothreonine
?
show the reaction diagram
-
-
-
-
-
D-serine
?
show the reaction diagram
D-serine
pyruvate + NH3
show the reaction diagram
D-threonine
2-oxobutanoate + NH3
show the reaction diagram
D-threonine
?
show the reaction diagram
-
-
-
-
?
L-serine
pyruvate + NH3
show the reaction diagram
S-serine
pyruvate + NH3
show the reaction diagram
-
-
-
-
?
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
D-serine
?
show the reaction diagram
D-serine
pyruvate + NH3
show the reaction diagram
L-serine
pyruvate + NH3
show the reaction diagram
-
-
-
-
?
S-serine
pyruvate + NH3
show the reaction diagram
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AMP
-
activates
ATP
-
activation at low concentrations, inhibition at high concentrations
CMP
-
activates
IMP
-
activates
pyridoxal 5'-phosphate
UMP
-
activates
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
-
the addition of Mn2+ induces a maximal recovery of 20% of the original activity in the EDTA-treated enzyme
additional information
-
no activation of the EDTA-treated enzyme is obtained after the addition of other divalent cations such as Mg2+, Ca2+, Cu2+ and Ni2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alkylamines
-
inactivation via a transimination of pyridoxal 5'-phosphate
Aminooxyacetate
-
-
ATP
-
activation at low concentrations, inhibition at high concentrations
DL-2,3-Diaminopropionic acid
-
-
DL-O-methyl serine
-
-
glycine
-
-
hydroxylamine
L-2,3-diaminopropionic acid
-
-
L-alanine
-
-
L-serine
-
-
N-ethylmaleimide
-
5 mM, DsdSC activity with pyridoxal 5'-phosphate 19.5%, without 8.7%
O-methylserine
-
competitive
phenylhydrazin
-
5 mM, DsdSC activity with pyridoxal 5'-phosphate 97.1%, without 4.2%
Sodium cyanoborohydride
-
5 mM, DsdSC activity with pyridoxal 5'-phosphate 20.9%, without 12.1%
Tris
-
inactivation is prevented by the presence of sufficient K+ or NH4+ and less effectively by Na+ or pyridoxal 5'-phosphate
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyridoxal 5'-deoxymethylenephosphonate
-
can substitute for pyridoxal 5'-phosphate, the enzyme exhibits 35-40% of the activity of the native enzyme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1 - 1.45
beta-Chloro-D-alanine
2.45
beta-Cl-D-alanine
-
EDTA-treated + 2.5 microM ZnCl2
0.63 - 0.88
D-allo-Thr
0.033
D-allo-threonine
-
pH not specified in the publication, 30C, wild-type
1.1 - 4.9
D-allothreonine
0.086 - 2.8
D-Ser
0.1 - 8.5
D-serine
0.083 - 16
D-Thr
0.039 - 0.312
D-threonine
4 - 20
L-Ser
12.3
L-serine
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.19 - 0.96
beta-Chloro-D-alanine
0.13
D-allo-threonine
Saccharomyces cerevisiae
-
pH not specified in the publication, 30C, wild-type
0.112 - 0.92
D-allothreonine
9.4 - 118
D-Ser
0.08 - 17
D-serine
0.95
D-Thr
Escherichia coli
-
pH 5.8, 25C
0.0512 - 0.43
D-threonine
1.8
L-allothreonine
Escherichia coli
-
pH 7.8, 25C
0.12 - 0.153
L-Ser
0.197
L-serine
Gallus gallus
-
-
17.2 - 19.6
L-Thr
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 90
D-serine
481
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.035
DL-2,3-Diaminopropionic acid
-
25C, pH 7.8
70
DL-O-methyl serine
-
25C, pH 7.8
4.9
glycine
-
25C, pH 7.8
0.33 - 0.46
Isoserine
0.09
L-2,3-diaminopropionic acid
-
25C, pH 7.8
17
L-alanine
-
25C, pH 7.8
3
L-serine
-
25C, pH 7.8
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0004
-
brain; liver
0.004
-
crude extract
0.0044
-
kidney
0.094
-
DEAE-Sepharose purified
0.52
-
ammonium sulfate precipitation
0.91
-
Mono Q purified
1.09
-
Hydroxyapatite purified
1.39
-
SuperSW3000 purified
2.12
-
selenomethionine labelled enzyme, pH 7.5, 37C
additional information
-
not detectable, activity of DSD in rats is compared to the activity of DSD in chicken
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8 - 8
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
-
pH 6.0: about 30% of maximal activity, pH 8.5: about 60% of maximal activity
6.5 - 9
-
pH 6.5: about 30% of maximal activity, pH 9.0: about 60% of maximal activity
6.5 - 11.5
-
in the range of pH 6.5-11.5 the enzyme has an activity between 50 and 100% with an maximum at pH 9
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
brain, cerebellum show about 6fold-higher activity than cerebrum
Manually annotated by BRENDA team
-
brain, cerebellum show about 6fold-higher activity than cerebrum
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Paraburkholderia xenovorans (strain LB400)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37300
-
analytical ultracentrifugation
40400
-
monomer 2, determined by SDS-PAGE
46000
-
gel filtration
48790
-
x * 48790, sequence analysis of peptides
50000
-
determined by SDS-PAGE
50060
-
calculated molecular mass
77000
-
molecular weight of the native enzyme, estimated by GC-LALLS
118000
-
dimer, determined by gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 48790, sequence analysis of peptides
heterodimer
-
1 * 40000 + 1 * 40400, SDS-PAGE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure is refined to 1.55 A resolution. Crystal structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain
-
enzyme complexed with 3-amino-2-hydroxypropionate
-
crystallized by the hanging-drop vapour-diffusion method using PEG 4000 and 2-propanol as precipitants. The crystal belong to space group P422, with unit-cell parameters a = 105.0, c = 81.89 A, and diffract to 2.09 A resolution
to 1.9 A resolution. In the active site pocket, a zinc ion that coordinates His347 and Cys349 is located near the PLP-Lys45 Schiff base. A theoretical model of the enzyme-D-serine complex suggests that the hydroxyl group of D-serine directly coordinates the zinc ion, and that the epsilon-NH2 group of Lys45 is a short distance from the substrate C-atom. The alpha-proton abstraction from D-serine by Lys45 and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity
-
homology model of Dsd. In this model, Dsd adopts a fold that is characteristic of type III pyridoxal-dependent enzymes, consisting of an alpha/beta TIM-barrel and a beta-sandwich domain at the N- and C-termini, respectively. Analysis of the Amide I and Amide III infrared bands reveals that the amounts of 24% alpha, 29% b and 47% unordered structures correlate well with those derived from the model, 25%, 29% and 46% respectively
-
both wild-type and selenomethionine labelled holoenzyme, to 2.4 and 1.9 A resolution, respectively. Significant electron density is not observed for the cofactor pyridoxal 5'-phosphate. Wild-type enzyme is in an open conformation while selenomethionine enzyme, crystallized in the presence of isoserine, is in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate. Thr166 may be involved in abstraction of proton from the Calpha atom of the substrate
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
6 h, 10% loss of activity
37
-
stable at and below, enzyme from mutant strain EM1610
50
-
enzyme from wild type strain EM1609 is stable, enzyme from mutant strain EM1610 is rapidly inactivated
54
-
1 min, dilute solution, complete loss of activity
55
-
enzyme from wild type strain is rapidly inactivated above
71.5
-
inactive form DsdR of the holoenzyme, obtained by reduction of the aldimine linkage tethering PLP to recombinant, tagged D-serine dehydratase by treatment with NaBH4
73.3
-
inactive form DsdR of the holoenzyme, obtained by reduction of the aldimine linkage tethering PLP to recombinant, tagged D-serine dehydratase by treatment with NaBH4, which is further treated with a protease in order to remove the amino-terminal purification tag
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
repeated freezing and thawing over a period of 1 or 2 months causes a 20-30% loss of activity
-
resolved completely by dialysis against L-Cys or D-Cys, reactivated by addition of pyridoxal 5'-phosphate
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0C, stable almost indefinitely
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
from chicken kidneys using a DEAE-Sepharose, a hydroxyapatite, a Mono Q HR5/5 and a TSKgel SuperSW3000 column
-
purified from k12 mutant C6
-
to homogeneity
-
using a Ni-chelating DEAE-TOYOPEARL and a gel permeation column, from 1 l culture 8 mg Dsd1p is obtained
-
using a Ni2+-chelating column and a DEAE-TOYOPEARL column
-
using Ni-NTA chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
expressed in Escherichia coli as a His-tagged fusion protein
-
expression in Escherichia coli
expression in Saccharomyces cerevisiae
-
into the pCR4-TOPO vector for sequencing
-
into the pET15b vector for expression in Escherichia coli BL21DE3 cells
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G279D
-
mutant G279D and G281D, loss of activity, the mutant enzymes form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. G279D has 225fold reduced cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 765fold. Mutant G281D has 50fold decreased cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 1970fold
G281D
-
mutant G279D and G281D, loss of activity, the mutant enzymes form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. G279D has 225fold reduced cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 765fold. Mutant G281D has 50fold decreased cofactor affinity, the ability to retain a cofactor:glycine complex is decreased 1970fold
C400A
-
dehydratase activity is completely abolished. Km (beta-chloro-D-alanine) decreased in native and Zn2+-depleted form compared to wild-type. kcat (beta-chloro-D-alanine) increased in native and Zn2+-depleted form compared to wild-type
H398A
-
dehydratase activity is completely abolished, kcat (D-serine) highly decreased. Km (beta-chloro-D-alanine) decreased in native and Zn2+-depleted form compared to wild-type. kcat (beta-chloro-D-alanine) increased in native and Zn2+-depleted form compared to wild-type
K57A
-
kcat (D-serine) decreased compared to wild-type, no reaction towards beta-chloro-D-alanine
Y203A
-
Km increased compared to wild-type, kcat decreased compared to wild-type. kcat ratio: 14% (wild-type 100%)
Y203D
-
Km increased compared to wild-type, kcat decreased compared to wild-type. kcat ratio: 0.57% (wild-type 100%)
Y203E
-
Km increased compared to wild-type, kcat decreased compared to wild-type. kcat ratio: 0.47% (wild-type 100%)
Y203F
-
Km increased compared to wild-type, kcat decreased compared to wild-type. kcat ratio: 25% (wild-type 100%)
Y203R
-
Km increased compared to wild-type, kcat decreased compared to wild-type. kcat ratio: 2.9% (wild-type 100%)
Y203S
-
Km increased compared to wild-type, kcat decreased compared to wild-type. kcat ratio: 14% (wild-type 100%)
additional information
-
reduction of the aldimine linkage tethering PLP to recombinant, tagged D-serine dehydratase by treatment with NaBH4 so as to yield an inactive form of the holoenzyme, DsdR, which is further treated with a protease in order to remove the amino-terminal purification tag. Fourier Transform infrared spectroscopic analysis reveals that both the reduced form DsdR and the reduced/detagged form DsdRD maintain the overall secondary structure of wild-type, but feature a significantly increased thermal stability. The observed Tm values for DsdR and for DsdRD shift to 71.5C and 73.3C, respectively, resulting in nearly 11 and 13 higher than the one measured for wild-type. Though catalytically inert, DsdRD retains the ability to enantioselectively bind its natural substrate
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the enzyme activity on D-serine decreases 20fold by EDTA treatment and recovers nearly completely by the addition of Zn2+
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
degradation
medicine
molecular biology
-
the dsdA gene is used as a selectable marker for transformation of Arabidopsis
pharmacology
-
the D-serine dehydratase gene is an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed
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