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Literature summary for 2.5.1.65 extracted from

  • 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 (2014), J. Bacteriol., 196, 3410-3420.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.5.1.65

Cloned(Commentary)

Cloned (Comment) Organism
gene cysK2 or Rv0848, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) Mycobacterium tuberculosis

Protein Variants

Protein Variants Comment Organism
additional information construction of truncated variant CysK2NT Mycobacterium tuberculosis
R243A site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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 Mycobacterium tuberculosis
0.044
-
 thiosulfate pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis
0.135
-
 O-phospho-L-serine dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis
0.214
-
 thiosulfate pH 7.0, 22°C, recombinant mutant R243A enzyme Mycobacterium tuberculosis
0.374
-
 thiosulfate dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis
0.485
-
 O-phospho-L-serine pH 7.0, 22°C, recombinant mutant R243A enzyme Mycobacterium tuberculosis
1.085
-
 O-phospho-L-serine dephosphorylation, pH 7.0, 22°C, recombinant mutant R243A enzyme Mycobacterium tuberculosis
1.086
-
 O-phospho-L-serine pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
O-phospho-L-serine + thiosulfate Mycobacterium tuberculosis 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 S-sulfocysteine + phosphate
-
 ?
O-phospho-L-serine + thiosulfate Mycobacterium tuberculosis H37Rv 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 S-sulfocysteine + phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Mycobacterium tuberculosis Q79FV4 gene cysK2 or Rv0848
-
Mycobacterium tuberculosis H37Rv Q79FV4 gene cysK2 or Rv0848
-

Purification (Commentary)

Purification (Comment) Organism
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 Mycobacterium tuberculosis

Reaction

Reaction Comment Organism Reaction ID
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 Mycobacterium tuberculosis
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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 Mycobacterium tuberculosis ?
-
 ?
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 Mycobacterium tuberculosis H37Rv ?
-
 ?
O-phospho-L-serine + thiosulfate
-
 Mycobacterium tuberculosis S-sulfocysteine + phosphate
-
 ?
O-phospho-L-serine + thiosulfate 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 Mycobacterium tuberculosis S-sulfocysteine + phosphate
-
 ?
O-phospho-L-serine + thiosulfate
-
 Mycobacterium tuberculosis H37Rv S-sulfocysteine + phosphate
-
 ?
O-phospho-L-serine + thiosulfate 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 Mycobacterium tuberculosis H37Rv S-sulfocysteine + phosphate
-
 ?

Subunits

Subunits Comment Organism
dimer in solution Mycobacterium tuberculosis

Synonyms

Synonyms Comment Organism
CysK2
-
 Mycobacterium tuberculosis
O-phospho-L-serine-dependent S-sulfocysteine synthase
-
 Mycobacterium tuberculosis
O-phosphoserine S-sulfocysteine synthase
-
 Mycobacterium tuberculosis
S-sulfocysteine synthase
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
21 22 assay at Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0067
-
 O-phospho-L-serine dephosphorylation, pH 7.0, 22°C, recombinant mutant R243A enzyme Mycobacterium tuberculosis
0.2
-
 O-phospho-L-serine dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis
0.457
-
 O-phospho-L-serine pH 7.0, 22°C, recombinant mutant R243A enzyme Mycobacterium tuberculosis
0.913
-
 O-phospho-L-serine pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Mycobacterium tuberculosis
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 Mycobacterium tuberculosis

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate dependent on Mycobacterium tuberculosis

General Information

General Information Comment Organism
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 Mycobacterium tuberculosis
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 Mycobacterium tuberculosis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
additional information
-
 additional information the specificity constant kcat/Km of CysK2 for thiosulfate is 40fold higher than for sulfide Mycobacterium tuberculosis
0.841
-
 O-phospho-L-serine pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis
0.942
-
 O-phospho-L-serine pH 7.0, 22°C, recombinant mutant R243A enzyme Mycobacterium tuberculosis
1.16
-
 O-phospho-L-serine dephosphorylation, pH 7.0, 22°C, recombinant wild-type enzyme Mycobacterium tuberculosis
3.97
-
 O-phospho-L-serine apparent second-order rate of the first half-reaction, pH 7.0, 21°C, recombinant wild-type enzyme Mycobacterium tuberculosis