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Literature summary extracted from

  • Xiao, J.Y.; Lee, J.Y.; Tokuhiro, S.; Nagataki, M.; Jarilla, B.R.; Nomura, H.; Kim, T.I.; Hong, S.J.; Agatsuma, T.
    Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target (2013), PLoS Negl. Trop. Dis., 7, e2548.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
2.7.3.4 pharmacology the enzyme is a candidate chemotherapeutic target Clonorchis sinensis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.3.4 gene CLF_107872, DNA and amino acid sequence determination and analysis, sequence comarisons and phylogenetic analysis, the coding region of the cDNA of D1D2 is cloned into the pMAL-c2 vector and expressed as maltose binding protein fusion protein in Escherichia coli strain TB1, quantitative real-time PCR enzyme expression analysis. Recombinant expression of N-terminally GST-tagged enzyme D1 and D2 mutants in Escherichia coli strain BL21(DE3) pLysS Clonorchis sinensis

Protein Variants

EC Number Protein Variants Comment Organism
2.7.3.4 H61A site-directed mutagenesis of a domain 2 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 H61A site-directed mutagenesis of TKD1D2 in D2 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 I60A site-directed mutagenesis of a domain 1 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 I60A site-directed mutagenesis of TKD1D2 in D1 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 I63A site-directed mutagenesis of a domain 2 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 I63A site-directed mutagenesis of TKD1D2 in D2 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 R58A site-directed mutagenesis of a domain 1 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 R58A site-directed mutagenesis of TKD1D2 in D1 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y84A site-directed mutagenesis of a domain 1 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y84A site-directed mutagenesis of TKD1D2 in D1 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y84R site-directed mutagenesis of a domain 1 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y84R site-directed mutagenesis of TKD1D2 in D1 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y87A site-directed mutagenesis of a domain 2 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y87A site-directed mutagenesis of TKD1D2 in D2 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y87R site-directed mutagenesis of a domain 2 residue, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis
2.7.3.4 Y87R site-directed mutagenesis of TKD1D2 in D2 region, the enzyme mutant shows altered kinetics and reduced activity compared to the wild-type enzyme Clonorchis sinensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.3.4 additional information
-
 additional information comparisons of wild-type and mutant enzyme kinetics, overview Clonorchis sinensis
2.7.3.4 0.35
-
 Taurocyamine recombinant domain D1, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 0.46
-
 ATP recombinant domain D1, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 0.48
-
 Taurocyamine recombinant domain D2, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 0.49
-
 Taurocyamine recombinant full-length enzyme, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 0.75
-
 ATP recombinant domain D2, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 0.78
-
 ATP recombinant full-length enzyme, pH 8.0, 25°C Clonorchis sinensis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.3.4 Mg2+ required Clonorchis sinensis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.3.4 39720
-
 sequence calculation, D2 domain Clonorchis sinensis
2.7.3.4 40570
-
 sequence calculation, D1 domain Clonorchis sinensis
2.7.3.4 42000
-
-
 Clonorchis sinensis
2.7.3.4 80000
-
-
 Clonorchis sinensis
2.7.3.4 80270
-
 sequence calculation, full-length enzyme Clonorchis sinensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.3.4 ATP + taurocyamine Clonorchis sinensis
-
 ADP + N-phosphotaurocyamine
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.3.4 Clonorchis sinensis G7YHA8 gene CLF_107872
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.3.4 recombinant enzyme coding region as maltose binding protein fusion protein from Escherichia coli strain TB1 by amylose affinity chromatography too homogeneity. Recombinant N-terminally GST-tagged enzyme D1 and D2 mutants from Escherichia coli strain BL21(DE3) pLysSby glutathione affinity chromatography, the GST-tag is cleaved off by thrombin treatment Clonorchis sinensis

Source Tissue

EC Number Source Tissue Comment Organism Textmining
2.7.3.4 egg intra-uterine Clonorchis sinensis
-
2.7.3.4 integument
-
 Clonorchis sinensis
-
2.7.3.4 intestine
-
 Clonorchis sinensis
-
2.7.3.4 additional information enzyme expression is distributed in locomotive and reproductive organs of adult worms. The enzyme is stably expressed in both the adult and metacercariae stages, quantitative real time PCR expression analysis, overview. Immunohistochemic analysis Clonorchis sinensis
-
2.7.3.4 seminal vesicle
-
 Clonorchis sinensis
-
2.7.3.4 sperm
-
 Clonorchis sinensis
-
2.7.3.4 sucker oral and ventral suckers Clonorchis sinensis
-
2.7.3.4 testis
-
 Clonorchis sinensis
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.3.4 ATP + taurocyamine
-
 Clonorchis sinensis ADP + N-phosphotaurocyamine
-
 ?
2.7.3.4 ATP + taurocyamine high substrate specificity for taurocyamine Clonorchis sinensis ADP + N-phosphotaurocyamine
-
 ?
2.7.3.4 additional information residues R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 participate in binding taurocyamine. Recombinant CsTK enzyme protein has low sensitivity and specificity toward Clonorchis sinensis and platyhelminth-infected human sera on ELISA Clonorchis sinensis ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.3.4 monomer 1 * 42000, SDS-PAGE, recombinant detagged D1 domain, SDS-PAGE, 1 * 80000, recombinant full-length enzyme, SDS-PAGE Clonorchis sinensis

Synonyms

EC Number Synonyms Comment Organism
2.7.3.4 CsTK
-
 Clonorchis sinensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.3.4 25
-
 assay at Clonorchis sinensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.3.4 4.5
-
 ATP recombinant domain D2, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 4.5
-
 Taurocyamine recombinant domain D2, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 17.78
-
 ATP recombinant full-length enzyme, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 17.78
-
 Taurocyamine recombinant full-length enzyme, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 22.59
-
 ATP recombinant domain D1, pH 8.0, 25°C Clonorchis sinensis
2.7.3.4 22.59
-
 Taurocyamine recombinant domain D1, pH 8.0, 25°C Clonorchis sinensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.3.4 8
-
 assay at Clonorchis sinensis

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.3.4 ATP
-
 Clonorchis sinensis

pI Value

EC Number Organism Comment pI Value Maximum pI Value
2.7.3.4 Clonorchis sinensis sequence calculation, D2 domain
-
 7.39
2.7.3.4 Clonorchis sinensis sequence calculation, full-length enzyme
-
 7.88
2.7.3.4 Clonorchis sinensis sequence calculation, D1 domain
-
 8.09

General Information

EC Number General Information Comment Organism
2.7.3.4 evolution the enzyme belongs to the highly conserved phosphagen (PK) kinase family of enzymes. The polypeptide is homologous to taurocyamine kinase (TK) of the invertebrate animals and consists of two contiguous domains. Clonorchis sinensis CsTK gene consists of 13 exons intercalated with 12 introns. This suggests an evolutionary pathway originating from an arginine kinase gene group, and distinguishes annelid taurocyamine kinase from the general creatine kinase phylogenetic group Clonorchis sinensis