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Literature summary for 1.1.1.50 extracted from

  • Hwang, C.C.; Hsu, C.N.; Huang, T.J.; Chiou, S.J.; Hong, Y.R.
    Interactions across the interface contribute the stability of homodimeric 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (2009), Arch. Biochem. Biophys., 490, 36-41.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
D249A, D249S, and D249K mutants are expressed in Escherichia coli BL21(DE3) cells Comamonas testosteroni

Protein Variants

Protein Variants Comment Organism
D249A the mutation leads to 2fold increased Km value compared to the wild type, the mutant shows increased retention time, suggesting a smaller molecule size than dimeric wild type enzyme Comamonas testosteroni
D249A mutation interrupts salt bridge between residues D249 and R167, secondary structure similar to wild-type. 30fold decrease in catalytic efficiency, decrease in melting temperature Comamonas testosteroni
D249K he mutation leads to 4fold increased Km value compared to the wild type, the mutant shows increased retention time, suggesting a smaller molecule size than dimeric wild type enzyme Comamonas testosteroni
D249K mutation interrupts salt bridge between residues D249 and R167, secondary structure similar to wild-type. 1.4fold decrease in catalytic efficiency, decrease in melting temperature Comamonas testosteroni
D249S the mutant has similar kinetic parameters to wild type enzyme Comamonas testosteroni
D249S mutation interrupts salt bridge between residues D249 and R167, secondary structure similar to wild-type. 1400fold decrease in catalytic efficiency, decrease in melting temperature Comamonas testosteroni

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00305
-
NAD+ mutant D249S, pH 10.2, 25°C Comamonas testosteroni
0.0038
-
NAD+ wild type enzyme, in 0.1 M CAPS buffer, at pH 10.2, at 25°C Comamonas testosteroni
0.0038
-
NAD+ wild-type, pH 10.2, 25°C Comamonas testosteroni
0.005
-
NAD+ mutant enzyme D249S, in 0.1 M CAPS buffer, at pH 10.2, at 25°C Comamonas testosteroni
0.009
-
NAD+ mutant enzyme D249K, in 0.1 M CAPS buffer, at pH 10.2, at 25°C Comamonas testosteroni
0.009
-
NAD+ mutant D249K, pH 10.2, 25°C Comamonas testosteroni
0.016
-
NAD+ mutant enzyme D249A, in 0.1 M CAPS buffer, at pH 10.2, at 25°C Comamonas testosteroni
0.016
-
NAD+ mutant D249A, pH 10.2, 25°C Comamonas testosteroni

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
52000
-
calculated from amino acid sequence Comamonas testosteroni

Organism

Organism UniProt Comment Textmining
Comamonas testosteroni P80702
-
-

Purification (Commentary)

Purification (Comment) Organism
metal-chelate chromatography Comamonas testosteroni

Renatured (Commentary)

Renatured (Comment) Organism
thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appear as a two-state transition and three-state transition, respectively Comamonas testosteroni

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
androsterone + NAD+
-
Comamonas testosteroni 5alpha-androstan-3,17-dione + NADH + H+
-
?
androsterone + NAD+
-
Comamonas testosteroni 5alpha-androstane-3,17-dione + NADH + H+
-
?

Subunits

Subunits Comment Organism
dimer x-ray crystallography Comamonas testosteroni
More concentration-dependent dimerization. The calculated molecular masses through gel filtration chromatography for wild-type, mutants D249A, D249K, and D249S at the concentration of 1 mg/ml are 52, 42, 43, and 50 kDa, and change to 47, 25, 26, and 34 kDa at 0.01 mg/ml, respectively Comamonas testosteroni

Synonyms

Synonyms Comment Organism
3alpha-HSD/CR
-
Comamonas testosteroni
3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
-
Comamonas testosteroni

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
47.2
-
melting temperature, mutant D249S Comamonas testosteroni
47.9
-
melting temperature, mutant D249A Comamonas testosteroni
49.41
-
melting temperature, mutant D249K Comamonas testosteroni
54
-
the melting temperature of the wild type enzyme is at 54.76°C Comamonas testosteroni
54.8
-
melting temperature, wild-type Comamonas testosteroni