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Literature summary for 1.14.99.14 extracted from

  • Nguyen, K.T.; Virus, C.; Guennewich, N.; Hannemann, F.; Bernhardt, R.
    Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone (2012), ChemBioChem, 13, 1161-1166.
    View publication on PubMed

Application

Application Comment Organism
synthesis because of its capacity to hydroxylate steroids, CYP106A2 is an interesting candidate for industrial production of steroids or their intermediates. CYP106A2 is known as a 15beta-hydroxylase, but also shows minor 11alpha-hydroxylase activity for progesterone. 11alpha-Hydroxyprogesterone is an important pharmaceutical compound with anti-androgenic and blood-pressure-regulating activity Priestia megaterium

Protein Variants

Protein Variants Comment Organism
A106T/A395I site-directed mutagenesis, the mutant shows 14.3fold higher regioselectivity for 11alpha-hydroxylation and 39.3fold increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
A106T/A395I/R409L site-directed mutagenesis, the mutant shows 12.6fold higher regioselectivity for 11alpha-hydroxylation and 108fold increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
A106T/A395W/G379K/R409L site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
A243V/A395I site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
A243V/A395W/G397K site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
A395I site-directed mutagenesis, the mutant shows 8.9fold higher 11alpha-hydroxylation activity compared to the wild-type enzyme Priestia megaterium
A395W/G397K site-directed mutagenesis, the mutant shows 11.5fold higher 11alpha-hydroxylation activity compared to the wild-type enzyme Priestia megaterium
D217V/A395I site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
M155I/F165L/A395I site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
additional information direction of the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site-directed mutagenesis, with aid of data from a homology model of CYP106A2 containing docked progesterone, together with site-directed mutagenesis of active site residues. Distributions of amounts of monohydroxylated progesterone products (15beta-,11alpha-, 9alpha-, and 6beta-hydroxyprogesterone) in vivo of wild-type and mutant enzymes, overview Priestia megaterium
T247V/A395I site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
T247W/A395W/G379K site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
T248V/A395I site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
T89N/A106T/A395I/R409L site-directed mutagenesis, the mutant shows higher regioselectivity for 11alpha-hydroxylation and increased catalytic efficiency compared to the wild-type enzyme Priestia megaterium
T89N/A395I site-directed mutagenesis, the mutant shows 11.8fold higher regioselectivity for 11alpha-hydroxylation and 24.4fold increased catalytic efficiency compared to the wild-type enzyme. The production of 15beta-hydroxyprogesterone decreases from 50.4% of the wild-type to 4.8% of mutant T89N/A395I enzyme, whereas that of 11alpha-hydroxyprogesterone increases from 27.7% to 80.9% Priestia megaterium

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.02
-
progesterone pH not specified in the publication, 30°C, mutant A243V/A395I Priestia megaterium
0.026
-
progesterone pH not specified in the publication, 30°C, mutant A243V/A395W/G397K Priestia megaterium
0.028
-
progesterone pH not specified in the publication, 30°C, mutant D217V/A395I Priestia megaterium
0.032
-
progesterone pH not specified in the publication, 30°C, mutants A395I and M155I/F165L/A395I Priestia megaterium
0.038
-
progesterone pH not specified in the publication, 30°C, mutant T247V/A395I Priestia megaterium
0.039
-
progesterone pH not specified in the publication, 30°C, mutant T247W/A395W/G379K Priestia megaterium
0.046
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395I/R409L Priestia megaterium
0.048
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395W/G379K/R409L Priestia megaterium
0.052
-
progesterone pH not specified in the publication, 30°C, mutant T248V/A395I Priestia megaterium
0.055
-
progesterone pH not specified in the publication, 30°C, mutant A395W/G397K Priestia megaterium
0.084
-
progesterone pH not specified in the publication, 30°C, mutant T89N/A395I Priestia megaterium
0.091
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395I Priestia megaterium
0.109
-
progesterone pH not specified in the publication, 30°C, mutant T89N/A106T/A395I/R409L Priestia megaterium
0.203
-
progesterone pH not specified in the publication, 30°C, wild-type Priestia megaterium

Localization

Localization Comment Organism GeneOntology No. Textmining
cytosol
-
Priestia megaterium 5829
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
47500
-
x * 47500, SDS-PAGE Priestia megaterium

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Priestia megaterium the enzyme is capable of converting useful pharmaceutical targets such as 3-oxo-DELTA4-steroids (e.g., testosterone, 11-deoxycorticosterone, androstenedione, and progesterone) mainly into their 15beta-hydroxy homologues. In addition, the enzyme hydroxylates progesterone to a minor extent at the 11alpha-, 9alpha-, and 6beta-positions ?
-
?
progesterone + AH2 + O2 Priestia megaterium
-
11alpha-hydroxyprogesterone + A + H2O
-
?

Organism

Organism UniProt Comment Textmining
Priestia megaterium Q06069
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the enzyme is capable of converting useful pharmaceutical targets such as 3-oxo-DELTA4-steroids (e.g., testosterone, 11-deoxycorticosterone, androstenedione, and progesterone) mainly into their 15beta-hydroxy homologues. In addition, the enzyme hydroxylates progesterone to a minor extent at the 11alpha-, 9alpha-, and 6beta-positions Priestia megaterium ?
-
?
additional information CYP106A2-catalyzed hydroxylations of progesterone at the 15beta-, 11alpha-, 9alpha-, and 6beta-positions Priestia megaterium ?
-
?
progesterone + AH2 + O2
-
Priestia megaterium 11alpha-hydroxyprogesterone + A + H2O
-
?

Subunits

Subunits Comment Organism
? x * 47500, SDS-PAGE Priestia megaterium

Synonyms

Synonyms Comment Organism
CYP106A2
-
Priestia megaterium

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Priestia megaterium

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.3
-
progesterone pH not specified in the publication, 30°C, wild-type Priestia megaterium
0.9
-
progesterone pH not specified in the publication, 30°C, mutant A243V/A395W/G397K Priestia megaterium
1.1
-
progesterone pH not specified in the publication, 30°C, mutant A243V/A395I Priestia megaterium
1.2
-
progesterone pH not specified in the publication, 30°C, mutant A395W/G397K Priestia megaterium
1.3
-
progesterone pH not specified in the publication, 30°C, mutant A395I Priestia megaterium
1.9
-
progesterone pH not specified in the publication, 30°C, mutant T247V/A395I Priestia megaterium
2.5
-
progesterone pH not specified in the publication, 30°C, mutant T89N/A106T/A395I/R409L Priestia megaterium
2.7
-
progesterone pH not specified in the publication, 30°C, mutant T247W/A395W/G379K Priestia megaterium
2.9
-
progesterone pH not specified in the publication, 30°C, mutant T89N/A395I Priestia megaterium
3.5
-
progesterone pH not specified in the publication, 30°C, mutant T248V/A395I Priestia megaterium
4.3
-
progesterone pH not specified in the publication, 30°C, mutant D217V/A395I Priestia megaterium
4.5
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395W/G379K/R409L Priestia megaterium
5
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395I Priestia megaterium
6.4
-
progesterone pH not specified in the publication, 30°C, mutant M155I/F165L/A395I Priestia megaterium
6.9
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395I/R409L Priestia megaterium

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.4
-
progesterone pH not specified in the publication, 30°C, wild-type Priestia megaterium
21.7
-
progesterone pH not specified in the publication, 30°C, mutant A395W/G397K Priestia megaterium
23.2
-
progesterone pH not specified in the publication, 30°C, mutant T89N/A106T/A395I/R409L Priestia megaterium
34.2
-
progesterone pH not specified in the publication, 30°C, mutant T89N/A395I Priestia megaterium
34.5
-
progesterone pH not specified in the publication, 30°C, mutant A243V/A395W/G397K Priestia megaterium
42
-
progesterone pH not specified in the publication, 30°C, mutants A395II Priestia megaterium
49.9
-
progesterone pH not specified in the publication, 30°C, mutant T247V/A395I Priestia megaterium
53.1
-
progesterone pH not specified in the publication, 30°C, mutant A243V/A395I Priestia megaterium
55.1
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395I Priestia megaterium
66.8
-
progesterone pH not specified in the publication, 30°C, mutant T248V/A395I Priestia megaterium
70.6
-
progesterone pH not specified in the publication, 30°C, mutant T247W/A395W/G379K Priestia megaterium
93.4
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395W/G379K/R409L Priestia megaterium
151.4
-
progesterone pH not specified in the publication, 30°C, mutant A106T/A395I/R409L Priestia megaterium
151.5
-
progesterone pH not specified in the publication, 30°C, mutant D217V/A395I Priestia megaterium
201.8
-
progesterone pH not specified in the publication, 30°C, mutant M155I/F165L/A395I Priestia megaterium