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 (Comment) | Organism |
---|---|
CA 7alpha-HSDH, recombinant overexpression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Clostridium sardiniense |
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 | 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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Acinetobacter calcoaceticus | - |
- |
- |
Bacteroides fragilis | - |
- |
- |
Clostridium sardiniense | G9FRD7 | - |
- |
Escherichia coli | - |
- |
- |
Pseudomonas sp. | - |
- |
- |
Stenotrophomonas maltophilia | - |
- |
- |
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 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 | 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 | 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 | 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 Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
completely stable for 108 h | Clostridium sardiniense |
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 | 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 | 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 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 |