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Literature summary for 1.1.1.159 extracted from
- Enzymatic routes for the synthesis of ursodeoxycholic acid (2014), J. Biotechnol., 191, 11-21.
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | the enzyme is useful in production of ursodeoxycholic acid, a secondary bile acid, which is used as a drug for the treatment of various liver diseases | Escherichia coli |
| synthesis | the enzyme is useful in production of ursodeoxycholic acid, a secondary bile acid, which is used as a drug for the treatment of various liver diseases | Pseudomonas sp. |
| synthesis | the enzyme is useful in production of ursodeoxycholic acid, a secondary bile acid, which is used as a drug for the treatment of various liver diseases | Bacteroides fragilis |
| synthesis | the enzyme is useful in production of ursodeoxycholic acid, a secondary bile acid, which is used as a drug for the treatment of various liver diseases | Stenotrophomonas maltophilia |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| CA 7alpha-HSDH, recombinant overexpression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Clostridium sardiniense |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| purified recombinant enzyme complexed with taurochenodeoxycholic acid and NADP+, sitting drop vapour diffusion method, mixing of 200 nl of 1.12 mM protein in 50 mM Tris-HCl, pH 8.0, 200 mM NaCl,and 5.6 mM of NADP+ and taurochenodeoxycholic acid, with 200 nl of reservoir solution containing 0.1 M HEPES, pH 7.5, and 25% PEG-3350, and equilibration against 0.03 ml reservoir solution, at 20°C, X-ray diffraction structure determination and analysis at 2.0 A resolution | Clostridium sardiniense |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | development and evaluation of a selected multi-step reaction system for the synthesis of ursodeoxycholic acid, separation of each step by isolation of the intermediates using ultrafiltration membranes, overview | Escherichia coli |
| additional information | development and evaluation of a selected multi-step reaction system for the synthesis of ursodeoxycholic acid, separation of each step by isolation of the intermediates using ultrafiltration membranes, overview | Pseudomonas sp. |
| additional information | development and evaluation of a selected multi-step reaction system for the synthesis of ursodeoxycholic acid, separation of each step by isolation of the intermediates using ultrafiltration membranes, overview | Bacteroides fragilis |
| additional information | development and evaluation of a selected multi-step reaction system for the synthesis of ursodeoxycholic acid, separation of each step by isolation of the intermediates using ultrafiltration membranes, overview | Stenotrophomonas maltophilia |
| R16G | site-directed mutagenesis, kcat and Km of the R16G mutant increase by more than 4times and 5times compared with wild-type values, respectively, while the catalytic efficiency (kcat/Km) of R16G mutant decreases by 17.26% compared to the wild-type enzyme. The increase in Km indicates that affinity of R16G mutant toward NADP+ becomes weak, while the cofactor NADP(H) dissociates more easily from the binding site resulting in the increase in kcat | Clostridium sardiniense |
| R194G | site-directed mutagenesis, the mutant shows slightly reduced catalytic efficiency compared with NADP+ compared to the wild-type enzyme | Clostridium sardiniense |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.34 | - |
NADP+ | wild-type enzyme, pH and temperature not specified in the publication | Clostridium sardiniense |  |
| 0.72 | - |
NADP+ | mutant R194G, pH and temperature not specified in the publication | Clostridium sardiniense | |
| 1.77 | - |
NADP+ | mutant R16G, pH and temperature not specified in the publication | Clostridium sardiniense | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| cholate + NAD+ | Escherichia coli | - |
3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | Pseudomonas sp. | - |
3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | Acinetobacter calcoaceticus | - |
3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | Bacteroides fragilis | - |
3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | Stenotrophomonas maltophilia | - |
3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Acinetobacter calcoaceticus | - |
- |
- |
| Bacteroides fragilis | - |
- |
- |
| Clostridium sardiniense | G9FRD7 | - |
- |
| Escherichia coli | - |
- |
- |
| Pseudomonas sp. | - |
- |
- |
| Stenotrophomonas maltophilia | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, anion exchange chromatography, and gel filtration | Clostridium sardiniense |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 70 | - |
pH and temperature not specified in the publication | Acinetobacter calcoaceticus |
| 70 | - |
pH and temperature not specified in the publication | Stenotrophomonas maltophilia |
| 70 | - |
pH and temperature not specified in the publication | Clostridium sardiniense |
| 900 | - |
pH and temperature not specified in the publication | Pseudomonas sp. |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| chenodeoxycholic acid + NAD+ | - |
Stenotrophomonas maltophilia | 7-oxolithocholic acid + NADH + H+ | - |
? | |
| cholate + NAD+ | - |
Escherichia coli | 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | - |
Pseudomonas sp. | 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | - |
Acinetobacter calcoaceticus | 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | - |
Bacteroides fragilis | 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| cholate + NAD+ | - |
Stenotrophomonas maltophilia | 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholanate + NADH + H+ | - |
r | |
| lithocholic acid + NADPH + H+ | presence of both 7alpha-HSDH and 7beta-HSDH (EC 1.1.1.201) in one organism allows epimerization by a single bacterium | Escherichia coli | ursodeoxycholic acid + NADP+ | - |
? | |
| lithocholic acid + NADPH + H+ | presence of both 7alpha-HSDH and 7beta-HSDH (EC 1.1.1.201) in one organism allows epimerization by a single bacterium | Pseudomonas sp. | ursodeoxycholic acid + NADP+ | - |
? | |
| lithocholic acid + NADPH + H+ | presence of both 7alpha-HSDH and 7beta-HSDH (EC 1.1.1.201) in one organism allows epimerization by a single bacterium | Bacteroides fragilis | ursodeoxycholic acid + NADP+ | - |
? | |
| lithocholic acid + NADPH + H+ | presence of both 7alpha-HSDH and 7beta-HSDH (EC 1.1.1.201) in one organism allows epimerization by a single bacterium | Stenotrophomonas maltophilia | ursodeoxycholic acid + NADP+ | - |
? | |
| taurochenodeoxycholic acid + NADP+ | - |
Clostridium sardiniense | ? + NADPH + H+ | - |
r | |
| ursodeoxycholic acid + NAD+ | - |
Stenotrophomonas maltophilia | 7-oxolithocholic acid + NADH + H+ | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| tetramer | the enzyme possesses the typical alpha/beta folding pattern. The residues Glu258, Gln255 and Thr253 in one subunit form hydrogen bonds with the residues His211/Ser207, Ser207, Pro151 in the other subunit, respectively | Clostridium sardiniense |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| 7alpha-HSDH | - |
Clostridium sardiniense |
| 7alpha-hydroxysteroid dehydrogenase | - |
Escherichia coli |
| 7alpha-hydroxysteroid dehydrogenase | - |
Pseudomonas sp. |
| 7alpha-hydroxysteroid dehydrogenase | - |
Acinetobacter calcoaceticus |
| 7alpha-hydroxysteroid dehydrogenase | - |
Bacteroides fragilis |
| 7alpha-hydroxysteroid dehydrogenase | - |
Stenotrophomonas maltophilia |
| CA 7alpha-HSDH | - |
Clostridium sardiniense |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | - |
completely stable for 108 h | Clostridium sardiniense |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 13.76 | - |
NADP+ | wild-type enzyme, pH and temperature not specified in the publication | Clostridium sardiniense | |
| 28.66 | - |
NADP+ | mutant R194G, pH and temperature not specified in the publication | Clostridium sardiniense | |
| 58.55 | - |
NADP+ | mutant R16G, pH and temperature not specified in the publication | Clostridium sardiniense | |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NAD+ | NAD+-dependent | Escherichia coli | |
| NAD+ | NAD+-dependent | Pseudomonas sp. | |
| NAD+ | NAD+-dependent | Acinetobacter calcoaceticus | |
| NAD+ | NAD+-dependent | Bacteroides fragilis | |
| NAD+ | NAD+-dependent | Stenotrophomonas maltophilia | |
| NADP+ | cofactor binding site structure and active state structure of the NADP+ bound to the enzyme and enzyme mutants, overview. Residue Arg38 can form the stable cation-Pi interaction with the adenine ring of NADP+, and the cation-Pi interaction and hydrogen bonds between Arg38 and NADP+ have a significant anchor effect on the cofactor binding to CA 7alpha-HSDH. Residues Arg16, Arg38 and Arg194 mediate the cofactor specificity and recognition in right orientation | Clostridium sardiniense | |
| NADPH | - |
Clostridium sardiniense | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | 7alpha-HSDHs is a member of SDR superfamily and belongs to the classical subfamily with the cofactor binding site sequence motif TA/G/SxxxGIG and the active site sequence motif YxxxK (x = any amino acid residue) | Clostridium sardiniense |
| additional information | molecular dynamics simulation of cofactor NADPH and taurochenodeoxycholic acid bound to the enzyme, overview. The C-terminal structure of CA 7alpha-HSDH does not directly surround the substrate-binding pocket but stretch outward. The Ser-Tyr-Lys triad is well conserved in other 7alpha-HSDHs but not in the CA 7alpha-HSDH | Clostridium sardiniense |
| physiological function | the enzyme CA 7alpha-HSDH is responsible for reversibly catalysing the oxidation of C7alpha-oriented hydroxyl of the steroid nucleus in the bile acid metabolism | Clostridium sardiniense |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 33.18 | - |
NADP+ | mutant R16G, pH and temperature not specified in the publication | Clostridium sardiniense | |
| 39.78 | - |
NADP+ | mutant R194G, pH and temperature not specified in the publication | Clostridium sardiniense | |
| 40.1 | - |
NADP+ | wild-type enzyme, pH and temperature not specified in the publication | Clostridium sardiniense | |
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