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O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
ping-pong bi-bi mechanism
-
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
ping-pong bi-bi mechanism
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
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
-
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
active site structure, modeling of substrate binding at the active site with Arg297 being crucial for activity
-
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
catalytic cycle of CysK2 and related cysteine synthases, catalytic raction mechanism of enzyme CysK2 via formation of the enzyme-aminoacrylate intermediate, accompanied by the release of a phosphate ion, commonly observed in the class of pyridoxal 5'-phosphate-dependent enzymes, overview
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
ping-pong bi-bi mechanism, the active site of ApOPSS contains pyridoxal 5'-phosphate linked to lysine 127 as an internal Schiff base. Binding of the primary substrate O-phospho-L-serine displaces the lysine and forms an external Schiff base, initiating the first half-reaction that yields an alpha-aminoacrylate intermediate linked to pyridoxal 5'-phosphate. The second half-reaction involves the addition of a secondary substrate to the alpha-aminoacrylate intermediate and generates an external Schiff base with cysteine. The active-site lysine reacts with this external Schiff base, releasing cysteine and regenerating the internal Schiff base with K127. When other nucleophiles are used instead of sulfide, enzyme ApOPSS produces the corresponding non-natural amino acid
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
ping-pong bi-bi mechanism
-
-
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
ping-pong bi-bi mechanism, the active site of ApOPSS contains pyridoxal 5'-phosphate linked to lysine 127 as an internal Schiff base. Binding of the primary substrate O-phospho-L-serine displaces the lysine and forms an external Schiff base, initiating the first half-reaction that yields an alpha-aminoacrylate intermediate linked to pyridoxal 5'-phosphate. The second half-reaction involves the addition of a secondary substrate to the alpha-aminoacrylate intermediate and generates an external Schiff base with cysteine. The active-site lysine reacts with this external Schiff base, releasing cysteine and regenerating the internal Schiff base with K127. When other nucleophiles are used instead of sulfide, enzyme ApOPSS produces the corresponding non-natural amino acid
-
-
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
catalytic cycle of CysK2 and related cysteine synthases, catalytic raction mechanism of enzyme CysK2 via formation of the enzyme-aminoacrylate intermediate, accompanied by the release of a phosphate ion, commonly observed in the class of pyridoxal 5'-phosphate-dependent enzymes, overview
-
-
O-phospho-L-serine + hydrogen sulfide = L-cysteine + phosphate
-
-
-
-
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3-chloro-L-alanine + hydrogen sulfide
?
-
-
-
-
ir
3-chloro-L-alanine + sulfide
?
-
heat-labile substrate, 173% of activity compared with O-acetyl-L-serine as substrate
-
-
?
L-azaserine + hydrogen sulfide
?
-
O-phospho-L-serine is a heat-stable substrate
-
-
ir
L-azaserine + sulfide
?
-
same activity as with O-acetyl-L-serine as substrate
-
-
?
L-cysteine + dithiothreitol
S-(2,3-hydroxy-4-thiobutyl)-L-cysteine + sulfide
-
OASS has a high activity in the L-cysteine desulfurization reaction
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
O-acetyl-L-serine + sulfide
L-cysteine + acetic acid
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
O-phospho-L-serine + sulfide
L-cysteine + phosphate
-
heat-stabile substrate, 219% of activity compared with O-acetyl-L-serine as substrate, best substrate at pH 6.7 and 60°C, formation of an alpha-aminoacrylate intermediate between O-phospho-L-serine and pyridoxal 5-phosphate
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
O3-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
-
?
additional information
?
-
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
-
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
low activity
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
enzyme is involved in L-cysteine biosynthesis, pathway overview
-
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ir
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
O-acetyl-L-serine is a heat-labile substrate
-
-
ir
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
low activity
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
-
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
-
-
?
O-acetyl-L-serine + sulfide
L-cysteine + acetic acid
-
-
-
-
?
O-acetyl-L-serine + sulfide
L-cysteine + acetic acid
-
heat-labile substrate
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
best substrate
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
enzyme is involved in L-cysteine biosynthesis
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
enzyme is involved in L-cysteine biosynthesis, pathway overview
-
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ir
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
O-phospho-L-serine is a heat-stable substrate
-
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ir
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
residue Arg297 near the entrance of the active site and is important for O-phospho-L-serine substrate recognition
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
residue Arg297 near the entrance of the active site and is important for O-phospho-L-serine substrate recognition
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
best substrate
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
-
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
-
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
-
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
CysK2 utilizes O-phospho-L-serine and thiosulfate as acceptor and preferred sulfur donor substrates in a pyridoxal 5'-phosphate-dependent reaction resulting in the formation of S-sulfocysteine
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-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
-
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
CysK2 utilizes O-phospho-L-serine and thiosulfate as acceptor and preferred sulfur donor substrates in a pyridoxal 5'-phosphate-dependent reaction resulting in the formation of S-sulfocysteine
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?
additional information
?
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biosynthesis of L-cysteine
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-
?
additional information
?
-
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L-cysteine biosynthesis
-
-
?
additional information
?
-
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L-cysteine biosynthesis
-
-
?
additional information
?
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enzyme with cystathionine beta-synthase and O-acetylserine sulfhydrylase activity in vitro, OASS has also L-serine sulfhydrylation and S-sulfo-L-cysteine synthesis activity
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?
additional information
?
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not: 3-chloro-D-alanine, 3-cyano-L-alanine, O-benzyl-L-serine, O-tert-butyl-L-serine, O-phospho-D-serine, O-succinyl-L-homoserine, L-homoserine
-
-
?
additional information
?
-
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substrate specificity, no activity with 3-chloro-D-alanine, 3-cyano-L-alanine, O-benzyl-L-serine, O-tert-butyl-L-serine, O-phospho-D-serine, O-succinyl-L-homoserine, and L-homoserine
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-
?
additional information
?
-
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the enzyme also shows low L-cystathionine forming activity
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-
?
additional information
?
-
the enzyme also catalyzes the reaction of EC 2.5.1.47, cysteine synthase
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-
?
additional information
?
-
OPSS is able to catalyze the synthetic reactions of various unnatural amino acids from OPS and nucleophiles that can substitute for sulfide
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-
?
additional information
?
-
the enzyme is also active with O-acetyl-L-serine, cf. EC 2.5.1.47. When other nucleophiles are used instead of sulfide, enzyme ApOPSS produces the corresponding non-natural amino acid, when thiosulfate is used as nucleophile, for example, S-sulfocysteine is produced. In absence of sulfide, the primary substrate reacts with to pyridoxal 5'-phosphate in enzyme OPSS to yield an alpha-aminoacrylate intermediate, which is formed through an external Schiff base with the elimination of phosphate or acetate, the intermediate is finally degraded to pyruvate and pyridoxal 5'-phosphate by a water molecule without a nucleophile
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-
?
additional information
?
-
OPSS is able to catalyze the synthetic reactions of various unnatural amino acids from OPS and nucleophiles that can substitute for sulfide
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-
?
additional information
?
-
the enzyme is also active with O-acetyl-L-serine, cf. EC 2.5.1.47. When other nucleophiles are used instead of sulfide, enzyme ApOPSS produces the corresponding non-natural amino acid, when thiosulfate is used as nucleophile, for example, S-sulfocysteine is produced. In absence of sulfide, the primary substrate reacts with to pyridoxal 5'-phosphate in enzyme OPSS to yield an alpha-aminoacrylate intermediate, which is formed through an external Schiff base with the elimination of phosphate or acetate, the intermediate is finally degraded to pyruvate and pyridoxal 5'-phosphate by a water molecule without a nucleophile
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-
?
additional information
?
-
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enzyme is involved in an O-phosphoserine based cysteine biosynthesis pathway in Mycobacterium tuberculosis that is independent of both O-acetylserine and the sulphate reduction pathway
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-
?
additional information
?
-
enzyme is involved in an O-phosphoserine based cysteine biosynthesis pathway in Mycobacterium tuberculosis that is independent of both O-acetylserine and the sulphate reduction pathway
-
-
?
additional information
?
-
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O-acetylserine is not a substrate. Enzyme does not catalyze the reaction of EC 2.5.1.47, O-acetylserine sulfhydrolases
-
-
?
additional information
?
-
O-acetylserine is not a substrate. Enzyme does not catalyze the reaction of EC 2.5.1.47, O-acetylserine sulfhydrolases
-
-
?
additional information
?
-
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specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine. Specificity of CysM for this physiological sulfide equivalent, sulfur carrier protein CysO-COSH, is more than 3 orders of magnitude greater than that for bisulfide. CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer
-
-
?
additional information
?
-
the enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal 5'-phosphate-dependent enzyme family. Enzyme CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates, the specificity constant kcat/Km of CysK2 for thiosulfate is 40fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. No significant activity with O-acetyl-L-serine, Asp, Val, Gln, Glu, Ser, Asn, Cys, Ser, Leu, homocysteine, ketoacids such as pyruvate and 2-oxoglutarate, amino acid precursors like 3-phosphoglycerate and succinate, and derivatives like N-acetylcysteine and diaminopimelic acid
-
-
?
additional information
?
-
-
the enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal 5'-phosphate-dependent enzyme family. Enzyme CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates, the specificity constant kcat/Km of CysK2 for thiosulfate is 40fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. No significant activity with O-acetyl-L-serine, Asp, Val, Gln, Glu, Ser, Asn, Cys, Ser, Leu, homocysteine, ketoacids such as pyruvate and 2-oxoglutarate, amino acid precursors like 3-phosphoglycerate and succinate, and derivatives like N-acetylcysteine and diaminopimelic acid
-
-
?
additional information
?
-
enzyme is involved in an O-phosphoserine based cysteine biosynthesis pathway in Mycobacterium tuberculosis that is independent of both O-acetylserine and the sulphate reduction pathway
-
-
?
additional information
?
-
O-acetylserine is not a substrate. Enzyme does not catalyze the reaction of EC 2.5.1.47, O-acetylserine sulfhydrolases
-
-
?
additional information
?
-
the enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal 5'-phosphate-dependent enzyme family. Enzyme CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates, the specificity constant kcat/Km of CysK2 for thiosulfate is 40fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. No significant activity with O-acetyl-L-serine, Asp, Val, Gln, Glu, Ser, Asn, Cys, Ser, Leu, homocysteine, ketoacids such as pyruvate and 2-oxoglutarate, amino acid precursors like 3-phosphoglycerate and succinate, and derivatives like N-acetylcysteine and diaminopimelic acid
-
-
?
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O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
additional information
?
-
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
-
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
enzyme is involved in L-cysteine biosynthesis, pathway overview
-
-
ir
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
enzyme is involved in L-cysteine biosynthesis
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
enzyme is involved in L-cysteine biosynthesis, pathway overview
-
-
ir
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
CysK2 utilizes O-phospho-L-serine and thiosulfate as acceptor and preferred sulfur donor substrates in a pyridoxal 5'-phosphate-dependent reaction resulting in the formation of S-sulfocysteine
-
-
?
O-phospho-L-serine + thiosulfate
S-sulfocysteine + phosphate
CysK2 utilizes O-phospho-L-serine and thiosulfate as acceptor and preferred sulfur donor substrates in a pyridoxal 5'-phosphate-dependent reaction resulting in the formation of S-sulfocysteine
-
-
?
additional information
?
-
-
biosynthesis of L-cysteine
-
-
?
additional information
?
-
-
L-cysteine biosynthesis
-
-
?
additional information
?
-
-
L-cysteine biosynthesis
-
-
?
additional information
?
-
-
enzyme is involved in an O-phosphoserine based cysteine biosynthesis pathway in Mycobacterium tuberculosis that is independent of both O-acetylserine and the sulphate reduction pathway
-
-
?
additional information
?
-
enzyme is involved in an O-phosphoserine based cysteine biosynthesis pathway in Mycobacterium tuberculosis that is independent of both O-acetylserine and the sulphate reduction pathway
-
-
?
additional information
?
-
enzyme is involved in an O-phosphoserine based cysteine biosynthesis pathway in Mycobacterium tuberculosis that is independent of both O-acetylserine and the sulphate reduction pathway
-
-
?
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3-chloro-D-alanine
-
18% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
3-Cyano-L-alanine
-
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
-
25°C, 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
-
25°C, 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
-
25°C, 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
-
25°C, 10 min, 20% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 27% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
FeCl3
-
25°C, 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
-
25°C, 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
-
25°C, 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
-
36% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
O-tert-butyl-L-serine
-
49% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Pb(CH3COO)2
-
25°C, 10 min, 95% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 88% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
Pb2+
-
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
-
25°C, 10 min, 23% inhibition of the O-phospho-L-serine sulfhydrylation reaction, 25% inhibition of the O-acetyl-L-serine sulfhydrylation reaction
additional information
-
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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0.25 - 12.5
hydrogen sulfide
21 - 28
O-acetyl-L-serine
0.135 - 250
O-phospho-L-serine
0.044 - 0.374
thiosulfate
additional information
additional information
-
0.25
hydrogen sulfide
-
pH 7.6, 60°C, with O-acetyl-L-serine
0.39
hydrogen sulfide
pH 7.5, 80°C, wild-type enzyme, with O-acetyl-L-serine
1.6
hydrogen sulfide
pH 7.5, 80°C, mutant F225A, with O-phospho-L-serine
3.8
hydrogen sulfide
pH 7.5, 80°C, wild-type enzyme, with O-phospho-L-serine
5
hydrogen sulfide
-
pH 7.6, 60°C, with O-phospho-L-serine
12.5
hydrogen sulfide
-
pH 7.6, 85°C, with O-phospho-L-serine
21
O-acetyl-L-serine
-
60°C
21
O-acetyl-L-serine
-
pH 7.6, 60°C
28
O-acetyl-L-serine
-
pH 6.7
0.135
O-phospho-L-serine
dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme
0.485
O-phospho-L-serine
pH 7.0, 22°C, recombinant mutant R243A enzyme
1.085
O-phospho-L-serine
dephosphorylation, pH 7.0, 22°C, recombinant mutant R243A enzyme
1.086
O-phospho-L-serine
pH 7.0, 22°C, recombinant wild-type enzyme
200
O-phospho-L-serine
-
60°C
200
O-phospho-L-serine
-
pH 7.6, 60°C
250
O-phospho-L-serine
-
85°C
250
O-phospho-L-serine
-
pH 7.6, 85°C
0.2
Sulfide
-
below, pH 6.7
0.25
Sulfide
-
60°C, O-acetyl-L-serine sulfhydrylation reaction
5
Sulfide
-
60°C, O-phospho-L-serine sulfhydrylation reaction
12.5
Sulfide
-
85°C, O-phospho-L-serine sulfhydrylation reaction
0.044
thiosulfate
pH 7.0, 22°C, recombinant wild-type enzyme
0.214
thiosulfate
pH 7.0, 22°C, recombinant mutant R243A enzyme
0.374
thiosulfate
dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme
additional information
additional information
-
-
-
additional information
additional information
-
kinetics
-
additional information
additional information
-
ping-pong bi-bi mechanism
-
additional information
additional information
steady-state kinetics, 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
-
additional information
additional information
stopped-flow and Michaelis-Menten kinetic analysis, overview. The amino acrylate reaction intermediate is not stable and decomposes with a pseudo-first-order rate constant kobs of 0.12/s
-
additional information
additional information
-
stopped-flow and Michaelis-Menten kinetic analysis, overview. The amino acrylate reaction intermediate is not stable and decomposes with a pseudo-first-order rate constant kobs of 0.12/s
-
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metabolism
two biosynthetic routes to L-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), are described in Mycobacterium tuberculosis, and a third putative sulfhydrylase in this pathogen, CysK2, is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine, cysteine synthasis pathways overview
metabolism
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two biosynthetic routes to L-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), are described in Mycobacterium tuberculosis, and a third putative sulfhydrylase in this pathogen, CysK2, is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine, cysteine synthasis pathways overview
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physiological function
the enzyme synthesizes S-sulfocysteine, the can also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy
physiological function
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the enzyme synthesizes S-sulfocysteine, the can also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy
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additional information
Intramolecular electrostatic interaction of enzyme OPSS, overview
additional information
three-dimensional structure analysis of enzyme ApOPSS in complex with aminoacrylate intermediate formed from pyridoxal 5'-phosphate with O-phospho-L-serine or the enzyme in complex with L-cysteine, and structure comparisons, molecular docking simulation using the structure of PDB ID 3VSA as the wild-type enzyme ApOPSS, overview
additional information
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Intramolecular electrostatic interaction of enzyme OPSS, overview
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additional information
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three-dimensional structure analysis of enzyme ApOPSS in complex with aminoacrylate intermediate formed from pyridoxal 5'-phosphate with O-phospho-L-serine or the enzyme in complex with L-cysteine, and structure comparisons, molecular docking simulation using the structure of PDB ID 3VSA as the wild-type enzyme ApOPSS, overview
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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
hanging-drop vapor-diffusion method
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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 295°C, 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
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, 25°C, 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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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
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
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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
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K204A
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to improve crystallization of CysM alone, a putative surface residue in CysM (Lys204) is mutated to alanine using site-directed mutagenesis
R243A
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
K204A
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to improve crystallization of CysM alone, a putative surface residue in CysM (Lys204) is mutated to alanine using site-directed mutagenesis
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R220A
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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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R297A
0.3% of wild-type activity
R297A
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site-directed mutagenesis, highly reduced activity with phospho-L-serine compared to the wild-type enzyme
R220A
700fold lower activity with O-phospho-L-serine as substrate compared to the wild type enzyme
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
additional information
construction of truncated variant CysK2NT
additional information
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construction of truncated variant CysK2NT
additional information
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construction of truncated variant CysK2NT
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Mino, K.; Ishikawa, K.
Characterization of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1
J. Bacteriol.
185
2277-2284
2003
Aeropyrum pernix
brenda
Mino, K.; Oda, Y.; Ataka, M.; Ishikawa, K.
Crystallization and preliminary X-ray diffraction analysis of O-acetylserine sulfhydrylase from Aeropyrum pernix K1
Acta Crystallogr. Sect. D
59
338-340
2003
Aeropyrum pernix
brenda
Mino, K.; Ishikawa, K.
A novel O-phospho-L-serine sulfhydrylation reaction catalyzed by O-acetylserine sulfhydrylase from Aeropyrum pernix K1
FEBS Lett.
551
133-138
2003
Aeropyrum pernix
brenda
Oda, Y.; Mino, K.; Ishikawa, K.; Ataka, M.
Three-dimensional structure of a new enzyme, O-phosphoserine sulfhydrylase, involved in l-cysteine biosynthesis by a hyperthermophilic archaeon, Aeropyrum pernix K1, at 2.0 A resolution
J. Mol. Biol.
351
334-344
2005
Aeropyrum pernix
brenda
OLeary, S.E.; Jurgenson, C.T.; Ealick, S.E.; Begley, T.P.
O-Phospho-l-serine and the thiocarboxylated sulfur carrier protein CysO-COSH are substrates for CysM, a cysteine synthase from Mycobacterium tuberculosis
Biochemistry
47
11606-11615
2008
Mycobacterium tuberculosis
brenda
Agren, D.; Schnell, R.; Oehlmann, W.; Singh, M.; Schneider, G.
Cysteine synthase (CYSM) of Mycobacterium tuberculosis is an O-phosphoserine sulfhydrylase: Evidence for an alternative cysteine biosynthesis pathway in mycobacteria
J. Biol. Chem.
283
31567-31574
2008
Mycobacterium tuberculosis, Mycobacterium tuberculosis (P9WP53), Mycobacterium tuberculosis H37Rv (P9WP53)
brenda
Jurgenson, C.T.; Burns, K.E.; Begley, T.P.; Ealick, S.E.
Crystal structure of a sulfur carrier protein complex found in the cysteine biosynthetic pathway of Mycobacterium tuberculosis
Biochemistry
47
10354-10364
2008
Mycobacterium tuberculosis
brenda
Nakamura, T.; Kawai, Y.; Kunimoto, K.; Iwasaki, Y.; Nishii, K.; Kataoka, M.; Ishikawa, K.
Structural analysis of the substrate recognition mechanism in O-phosphoserine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1
J. Mol. Biol.
422
33-44
2012
Aeropyrum pernix (Q9YBL2)
brenda
Nakamura, T.; Asai, S.; Nakata, K.; Kunimoto, K.; Oguri, M.; Ishikawa, K.
Thermostability and reactivity in organic solvent of O-phospho-L-serine sulfhydrylase from hyperthermophilic archaeon Aeropyrum pernix K1
Biosci. Biotechnol. Biochem.
79
1280-1286
2015
Aeropyrum pernix (Q9YBL2), Aeropyrum pernix DSM 11879 (Q9YBL2)
brenda
Takeda, E.; Kunimoto, K.; Kawai, Y.; Kataoka, M.; Ishikawa, K.; Nakamura, T.
Role of F225 in O-phosphoserine sulfhydrylase from Aeropyrum pernix K1
Extremophiles
20
733-745
2016
Aeropyrum pernix (Q9YBL2), Aeropyrum pernix DSM 11879 (Q9YBL2)
brenda
Steiner, E.M.; Boeth, D.; Loessl, P.; Vilaplana, F.; Schnell, R.; Schneider, G.
CysK2 from Mycobacterium tuberculosis is an O-phospho-L-serine-dependent S-sulfocysteine synthase
J. Bacteriol.
196
3410-3420
2014
Mycobacterium tuberculosis (Q79FV4), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (Q79FV4)
brenda