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Information on EC 2.5.1.65 - O-phosphoserine sulfhydrylase and Organism(s) Mycobacterium tuberculosis and UniProt Accession P9WP53
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2.5.1.65 O-phosphoserine sulfhydrylaseIUBMB Comments
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.
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The taxonomic range for the selected organisms is: Mycobacterium tuberculosis
The expected taxonomic range for this enzyme is: Archaea, Bacteria
The expected taxonomic range for this enzyme is: Archaea, Bacteria
Synonyms
opss, apopss, o-phosphoserine sulfhydrylase, o-phospho-l-serine sulfhydrylase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
PATHWAY SOURCE
PATHWAYS
BRENDA
KEGG
-
-, -
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
37290-89-4
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
?
O3-phospho-L-serine + hydrogen sulfide
L-cysteine + phosphate
-
-
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
-
-
-
?
O-acetyl-L-serine + hydrogen sulfide
L-cysteine + acetate
-
-
-
?
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
-
-
?
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
?
-
-
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
?
-
-
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
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
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
-
-
?
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
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pyridoxal 5'-phosphate
bound via a covalent linkage to the side chain of Lys51
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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
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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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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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.025
O3-acetyl-L-serine
-
formation of the aminoacrylate intermediate
17
O3-phospho-L-serine
-
formation of the aminoacrylate intermediate
0.0067
O-phospho-L-serine
dephosphorylation, pH 7.0, 22°C, recombinant mutant R243A enzyme
0.2
O-phospho-L-serine
dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme
0.457
O-phospho-L-serine
pH 7.0, 22°C, recombinant mutant R243A enzyme
0.913
O-phospho-L-serine
pH 7.0, 22°C, recombinant wild-type enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.841
O-phospho-L-serine
pH 7.0, 22°C, recombinant wild-type enzyme
0.942
O-phospho-L-serine
pH 7.0, 22°C, recombinant mutant R243A enzyme
1.16
O-phospho-L-serine
dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme
3.97
O-phospho-L-serine
apparent second-order rate of the first half-reaction, pH 7.0, 21°C, recombinant wild-type enzyme
additional information
additional information
the specificity constant kcat/Km of CysK2 for thiosulfate is 40fold higher than for sulfide
-
additional information
additional information
-
the specificity constant kcat/Km of CysK2 for thiosulfate is 40fold higher than for sulfide
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
PDB
SCOP
CATH
UNIPROT
ORGANISM
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
R243A
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
additional information
additional information
construction of truncated variant CysK2NT
additional information
-
construction of truncated variant CysK2NT
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography and Superdex-200 gel filtration
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
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene cysK2 or Rv0848, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
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)
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
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)
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