Information on EC 2.5.1.65 - O-phosphoserine sulfhydrylase

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

EC NUMBER
COMMENTARY hide
2.5.1.65
-
RECOMMENDED NAME
GeneOntology No.
O-phosphoserine sulfhydrylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C-O bond cleavage
-
-
sulfhydrylation
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
-
Cysteine and methionine metabolism
-
-
Metabolic pathways
-
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Microbial metabolism in diverse environments
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Sulfur metabolism
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cysteine metabolism
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SYSTEMATIC NAME
IUBMB Comments
O-phospho-L-serine:hydrogen-sulfide 2-amino-2-carboxyethyltransferase
A pyridoxal-phosphate protein. The enzyme from Aeropyrum pernix acts on both O-phospho-L-serine and O3-acetyl-L-serine, in contrast with EC 2.5.1.47, cysteine synthase, which acts only on O3-acetyl-L-serine.
CAS REGISTRY NUMBER
COMMENTARY hide
37290-89-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-chloro-L-alanine + hydrogen sulfide
?
show the reaction diagram
-
-
-
-
ir
3-chloro-L-alanine + sulfide
?
show the reaction diagram
-
heat-labile substrate, 173% of activity compared with O-acetyl-L-serine as substrate
-
-
?
L-azaserine + hydrogen sulfide
?
show the reaction diagram
-
O-phospho-L-serine is a heat-stable substrate
-
-
ir
L-azaserine + sulfide
?
show the reaction diagram
-
same activity as with O-acetyl-L-serine as substrate
-
-
?
L-cysteine + dithiothreitol
S-(2,3-hydroxy-4-thiobutyl)-L-cysteine + sulfide
show the reaction diagram
-
OASS has a high activity in the L-cysteine desulfurization reaction
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
show the reaction diagram
O-acetyl-L-serine + sulfide
L-cysteine + acetic acid
show the reaction diagram
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
show the reaction diagram
O-phospho-L-serine + sulfide
L-cysteine + phosphate
show the reaction diagram
-
heat-stabile substrate, 219% of activity compared with O-acetyl-L-serine as substrate, best substrate at pH 6.7 and 60C, formation of an alpha-aminoacrylate intermediate between O-phospho-L-serine and pyridoxal 5-phosphate
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
show the reaction diagram
O3-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
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
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
show the reaction diagram
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
show the reaction diagram
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyridoxal 5'-phosphate
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-chloro-D-alanine
-
18% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
3-Cyano-L-alanine
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42% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Cd2+
-
slightly inhibites both O-acetyl-L-serine sulfhydrylation and O-phospho-L-serine sulfhydrylation
CdCl2
-
25C, 10 min, 26% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 15% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Co2+
-
strongly inhibits O-acetyl-L-serine sulfhydrylation, moderately inhibites O-phospho-L-serine sulfhydrylation
CoCl2
-
25C, 10 min, 61% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 98.8% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Cu2+
-
strongly inhibits O-acetyl-L-serine sulfhydrylation, moderately inhibites O-phospho-L-serine sulfhydrylation
CuCl2
-
25C, 10 min, 79% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 98.7% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Fe2+
-
slightly inhibites both O-acetyl-L-serine sulfhydrylation and O-phospho-L-serine sulfhydrylation
Fe3+
-
strongly inhibits O-acetyl-L-serine sulfhydrylation, slightly inhibites O-phospho-L-serine sulfhydrylation
FeCl2
-
25C, 10 min, 20% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 27% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
FeCl3
-
25C, 10 min, 25% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 96.1% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Hg2+
-
strongly inhibites both O-acetyl-L-serine sulfhydrylation and O-phospho-L-serine sulfhydrylation
HgCl2
-
25C, 10 min, 98.3% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 80% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Ni2+
-
strongly inhibits O-acetyl-L-serine sulfhydrylation, slightly inhibites O-phospho-L-serine sulfhydrylation
NiCl2
-
25C, 10 min, 15% inhibition of the O-phospho-L-serine sulfhydrylation reaction, almost complete inhibition of the O-acetyl-L-serine sulfhydrylation reaction
O-benzyl-L-serine
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36% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
O-tert-butyl-L-serine
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49% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Pb(CH3COO)2
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25C, 10 min, 95% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 88% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Pb2+
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strongly inhibites both O-acetyl-L-serine sulfhydrylation and O-phospho-L-serine sulfhydrylation
Zn2+
-
slightly inhibites both O-acetyl-L-serine sulfhydrylation and O-phospho-L-serine sulfhydrylation
ZnCl2
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25C, 10 min, 23% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 25% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
additional information
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no inhibition by O-phospho-D-serine, EDTA, 2-mercaptoethanol, DTT, NEM, PCMB, and Gd3+, while Ca2+, K+, Na+, Mn2+, and Mg2+ are poor inhibitors; no inhibition of the O-acetyl-L-serine sulfhydrylation reaction by O-phospho-D-serine
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
-
slightly activates the O-phospho-L-serine sulfhydrylation reaction
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.25 - 12.5
hydrogen sulfide
21 - 28
O-acetyl-L-serine
0.135 - 250
O-phospho-L-serine
0.2 - 12.5
Sulfide
0.044 - 0.374
thiosulfate
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
33 - 730
hydrogen sulfide
2.6 - 202
O-acetyl-L-serine
0.0067 - 14000
O-phospho-L-serine
0.025
O3-acetyl-L-serine
Mycobacterium tuberculosis
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formation of the aminoacrylate intermediate
17
O3-phospho-L-serine
Mycobacterium tuberculosis
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formation of the aminoacrylate intermediate
additional information
additional information
Aeropyrum pernix
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-
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
81 - 190
hydrogen sulfide
911
0.07 - 3.5
O-acetyl-L-serine
680
0.841 - 11
O-phospho-L-serine
1189
additional information
additional information
Mycobacterium tuberculosis
Q79FV4
the specificity constant kcat/Km of CysK2 for thiosulfate is 40fold higher than for sulfide
2
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1
-
mutant R297A, substrate O-phospho-L-serine
23
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pH 7.5, 80C
50.9
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pH 6.7, 60C, O-acetyl-L-serine sulfhydrylation
53.1
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mutant R297A, substrate O-acetyl-L-serine
54.5
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60C, O-acetyl-L-serine sulfhydrylation reaction, recombinant OASS
72
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wild-type enzyme, substrate O-acetyl-L-serine
245
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wild-type enzyme, substrate O-phospho-L-serine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3 - 8.1
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at 85C; dependent on the substrate, overview
7.5
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assay at
9
the pH optimum for the dephosphorylation reaction is around pH 9.0, most likely because the spontaneous hydrolysis of the aminoacrylate intermediate is faster at this pH value, resulting in an increased turnover rate in the absence of the sulfur donor
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
-
sulfhydrylation of 3-chloro-L-alanine; with substrate 3-chloro-L-alanine
85
-
assay at
90
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sulfhydrylation of O-phospho-L-serine; with substrate O-phospho-L-serine
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70580
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sedimentation equilibrium ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
an enzyme monomer consists of 3 domains, structure overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme ApOPSS in complex with aminoacrylate intermediate formed from pyridoxal 5'-phosphate with O-phospho-L-serine or enzyme in complex with L-cysteine, hanging drop vapor diffusion method, mixing of 10 mg/ml protein in 50 mM potassium phosphate, pH 7.5, containing 0.2 mM pyridoxal 5'-phosphate, 2 mM EDTA, and 2 mM TCEP-HCl, with reservoir solutions containing 0.1 M HEPES, pH 7.5, 27% v/v 2-propanol, 10-12% v/v PEG 4000, and 12 mM TCEP-HCl, X-ray diffraction structure determinatoion and analysis at 2.14-2.15 A resolution, structure modeling
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hanging-drop vapor-diffusion method; purified recombinant enzyme, optimization of crystallization method, hanging drop vapour diffusion method, enzyme in 50 mM potassium phosphate buffer pH 7.5, containing 0.2 mM pyridoxal 5'-phosphate and 0.05% sodium azide, 0.0015 ml of enzyme and reservoir solution, containing 0.1 M sodium cacodylate pH 7.4, 0.1 M sodium acetate and 30% v/v PEG 8000, are equilibrated against 0.5 ml of reservoir solution at 295C, 2 weeks, cryoprotection by 10% v/v glycerol, X-ray diffraction structure determination and analysis at 2.2 A resolution
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structures of the enzyme without acetate, the complex formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with O-phosphoserine, and the complex formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with O-acetylserine, to 2.1 A resolution. No significant difference is seen in the overall structure between the free and complexed forms of the enzyme. The side chains of T152, S153, and Q224 interact with the carboxylate of the substrate. The position of R297 is significantly unchanged in the complex of the K127A mutant with the external Schiff base, allowing enough space for an interaction with O-phosphoserine. The positively charged environment around the entrance of the active site including S153 and R297 is important for accepting negatively charged substrates
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wild-type and selennomethionine-labeled enzyme, sitting drop method, 20 mg/ml protein in solution with 0.1 mM pyridoxal 5'-phosphate, 3 mM 2-mercaptoethanol, reservoir solution contains 0.1 M sodium cacodylate, pH 7.0, 0.2 M sodium acetate, 21% w/v poylethylene glycol 8000, and 3 mM 2-mercaptoethanol, mixture of equal volumes of protein and reservoir solution of 0.0035 ml, equilibration against 0.1 ml reservoir solution, 25C, 2 weeks, crystallization of the selenomethinone enzyme at pH 6.0, multi-wave anomalous dispersion, X-ray diffraction structure determination and analysis at 2.0 A resolution
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2.1 A resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity; sitting drop vapour diffusion method, with 0.1 M Tris-HCl pH 7.25-7.5, 0.1 M K2HPO4, 4.3 M NaCl
the structure of the protein complex CysM-CysO is determined at 1.53 A resolution. The protein complex in the crystal structure is asymmetric with one CysO (sulfur carrier protein) protomer binding to one end of a CysM dimer. The structures of CysM is determined individually at 2.8 A resolution. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, reveal high conservation of active site residues, but residues in CysM responsible for CysO binding are not conserved
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.1
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6 h, 100C, 10% loss of activity
637385
6.7
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6 h, 100C, 10% loss of activity
637385
7.5
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6 h, 100C, 44% loss of activity
637385
8.5
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6 h, 100C, 89% loss of activity
637385
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 100
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purified recombinant His-tagged enzyme, 1 h, completely stable at
90
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50% of maximum activity with 3-chloro-L-alanine as substrate
100
-
6 h, pH 6.1 and 6.7: 10% loss of activity, pH 7.5: 44% loss of activity, pH 8.5: 89% loss of activity
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1,4-dioxane
N,N-dimethylformamide
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, enzyme solution, stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography and Superdex-200 gel filtration
recombinant enzyme from Escherichia coli; recombinant OASS
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recombinant His-tagged enzyme from Escherichia coli strain BL21 (DE3) pLysS by nickel affinity chromatography
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recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and desalting gel filtration, followed by cleavage of the His-tag using thrombin and tag elimination through another sequence of nickel affinity chromatography and gel filtration
recombinant OASS
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recombinant wild-type, selenomethinonine-labeled, and mutant R297A enzymes from Escherichia coli
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using Ni-NTA chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli as a His-tagged fusion protein
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expressed in Escherichia coli BL21(DE3) cells; expression in Escherichia coli
expression in Escherichia coli Rosetta (DE3)
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expression in Escherichia coli Rosetta (DE3), sequencing
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expression of wild-type, selenomethinonine-labeled, and mutant R297A enzymes in Escherichia coli
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gene cysK2 or Rv0848, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
overexpression in Escherichia coli; overexpression in Escherichia coli Rosetta (DE3)
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recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 (DE3) pLysS
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F225A
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site-directed mutagenesis, the Km value toward O-phospho-L-serine is not significantly different between the wild-type ApOPSS and the F225A mutant, the kcat value of the wild-type ApOPSS is 4.2fold higher toward O-phospho-L-serine and 15fold higher toward O-acetyl-L-erine than that of the F225A mutant, respectively. The mutation from phenylalanine to alanine at position 225 affects the catalytic activity, not substrate binding
K127A
-
mutant is inactive for cysteine synthesis and does not form the alpha-aminoacrylate intermediate
Q224A
-
0.04% of wild-type activity
R297E
-
11% of wild-type activity
R297K
-
0.2% of wild-type activity
S153A
-
0.4% of wild-type activity
S153T
-
0.2% of wild-type activity
T152A
-
0.2% of wild-type activity
T152S
-
71% of wild-type activity
T203A
-
36% of wild-type activity
T203M
-
0.3% of wild-type activity
F225A
-
site-directed mutagenesis, the Km value toward O-phospho-L-serine is not significantly different between the wild-type ApOPSS and the F225A mutant, the kcat value of the wild-type ApOPSS is 4.2fold higher toward O-phospho-L-serine and 15fold higher toward O-acetyl-L-erine than that of the F225A mutant, respectively. The mutation from phenylalanine to alanine at position 225 affects the catalytic activity, not substrate binding
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K204A
-
to improve crystallization of CysM alone, a putative surface residue in CysM (Lys204) is mutated to alanine using site-directed mutagenesis
R220A
700fold lower activity with O-phospho-L-serine as substrate compared to the wild type enzyme; significant loss in specificity for substrate O-phosphoserine. The purified R220A mutant shows an absorption spectrum identical to wild type CysM with an absorption band at 412 nm reflecting the Schiff base between Lys51 and PLP. Formation of the aminoacrylate intermediate from O-phospho-L-serine in the mutant is severely compromised, with an approximately 700fold slower rate
R243A
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
K204A
-
to improve crystallization of CysM alone, a putative surface residue in CysM (Lys204) is mutated to alanine using site-directed mutagenesis
-
R220A
-
significant loss in specificity for substrate O-phosphoserine. The purified R220A mutant shows an absorption spectrum identical to wild type CysM with an absorption band at 412 nm reflecting the Schiff base between Lys51 and PLP. Formation of the aminoacrylate intermediate from O-phospho-L-serine in the mutant is severely compromised, with an approximately 700fold slower rate
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R243A
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site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
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additional information