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Literature summary for 1.1.1.159 extracted from

  • Huang, B.; Zhao, Q.; Zhou, J.H.; Xu, G.
    Enhanced activity and substrate tolerance of 7alpha-hydroxysteroid dehydrogenase by directed evolution for 7-ketolithocholic acid production (2019), Appl. Microbiol. Biotechnol., 103, 2665-2674 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Clostridium sardiniense

Protein Variants

Protein Variants Comment Organism
C260S the mutant shows 2fold higher specific activity as compared to the wild type enzyme Clostridium sardiniense
L26M the mutant shows 1.26fold higher specific activity as compared to the wild type enzyme Clostridium sardiniense
Q255L the mutant shows 4.2fold higher specific activity as compared to the wild type enzyme Clostridium sardiniense
Q255L/C260S the mutant shows 6.5fold higher specific activity and exhibits 10fold higher and 14fold higher catalytic efficiencies toward chenodeoxycholic acid and NADP+, respectively, as compared to the wild type enzyme. The mutant also displays significantly enhanced tolerance in the presence of high concentrations of substrate compared to the wild type Clostridium sardiniense
Q255R the mutant shows 1.3fold higher specific activity as compared to the wild type enzyme Clostridium sardiniense

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0601
-
chenodeoxycholic acid mutant enzyme Q255L/C260S, at pH 8.0 and 25┬░C Clostridium sardiniense
0.0661
-
chenodeoxycholic acid wild type enzyme, at pH 8.0 and 25┬░C Clostridium sardiniense
0.1094
-
NADP+ mutant enzyme Q255L/C260S, at pH 8.0 and 25┬░C Clostridium sardiniense
0.3116
-
NADP+ wild type enzyme, at pH 8.0 and 25┬░C Clostridium sardiniense

Organism

Organism UniProt Comment Textmining
Clostridium sardiniense G9FRD7 formerly known as Clostridium sardiniense strain DSM599
-
Clostridium sardiniense DSM 1599 G9FRD7 formerly known as Clostridium sardiniense strain DSM599
-

Purification (Commentary)

Purification (Comment) Organism
Ni-IDA affinity column chromatography Clostridium sardiniense

Specific Activity [micromol/min/mg]

Specific Activity Minimum [┬Ámol/min/mg] Specific Activity Maximum [┬Ámol/min/mg] Comment Organism
5.7
-
wild type enzyme, at pH 8.0 and 25┬░C Clostridium sardiniense

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
chenodeoxycholic acid + NADP+
-
Clostridium sardiniense 7-ketolithocholic acid + NADPH + H+
-
r
chenodeoxycholic acid + NADP+
-
Clostridium sardiniense DSM 1599 7-ketolithocholic acid + NADPH + H+
-
r

Subunits

Subunits Comment Organism
? x * 31000, SDS-PAGE Clostridium sardiniense

Synonyms

Synonyms Comment Organism
NADP+-dependent 7alpha-HSDH
-
Clostridium sardiniense

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
25
-
-
Clostridium sardiniense

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.34
-
chenodeoxycholic acid wild type enzyme, at pH 8.0 and 25┬░C Clostridium sardiniense
3.66
-
NADP+ wild type enzyme, at pH 8.0 and 25┬░C Clostridium sardiniense
18.45
-
NADP+ mutant enzyme Q255L/C260S, at pH 8.0 and 25┬░C Clostridium sardiniense
21
-
chenodeoxycholic acid mutant enzyme Q255L/C260S, at pH 8.0 and 25┬░C Clostridium sardiniense

Cofactor

Cofactor Comment Organism Structure
NADP+ dependent on Clostridium sardiniense

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
11.8
-
NADP+ wild type enzyme, at pH 8.0 and 25┬░C Clostridium sardiniense
35.4
-
chenodeoxycholic acid wild type enzyme, at pH 8.0 and 25┬░C Clostridium sardiniense
168.6
-
NADP+ mutant enzyme Q255L/C260S, at pH 8.0 and 25┬░C Clostridium sardiniense
349.4
-
chenodeoxycholic acid mutant enzyme Q255L/C260S, at pH 8.0 and 25┬░C Clostridium sardiniense