Information on EC 1.14.15.22 - vitamin D 1,25-hydroxylase

cytochrome P450SU-1

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Streptomyces griseolus cytochrome P450SU-1

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vitamin D3 dihydroxylase

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Biosynthesis of secondary metabolites, Steroid biosynthesis

calcidiol + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2 = calcitriol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

(2)

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calciol + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ = calcidiol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

(1)

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KEGG

calciol,ferredoxin:oxygen oxidoreductase (1,25-hydroxylating)

A P-450 (heme-thiolate) enzyme found in the bacterium Streptomyces griseolus. cf. EC 1.14.14.24, vitamin D 25-hydroxylase and EC 1.14.15.18, calcidiol 1-monooxygenase.

1alpha,25-dihydroxyvitamin D3 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

1alpha,25(R),26-trihydroxyvitamin D3 + 1alpha,25(S),26-trihydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

Substrates: -
Products: reaction of mutant R73V/R84A, 25R- and 25S-enantiomers are found in a ratio of 5:1

?

1alpha-hydroxyvitamin D3 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

1alpha,25-dihydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

25-hydroxyvitamin D2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

1alpha,25-dihydroxyvitamin D2 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

737618

Substrates: -
Products: reaction of mutants R73A/R84A and R73V/R84A

?

25-hydroxyvitamin D2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

25,26-dihydroxyvitamin D2 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

737618

Substrates: -
Products: reaction of mutants R73A/R84A and R73V/R84A

?

25-hydroxyvitamin D3 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

1alpha,25-dihydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

calcidiol + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

calcitriol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

calciol + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+

calcidiol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

vitamin D2 + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+

25-hydroxyvitamin D2 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

737586, 737618

Substrates: -
Products: -

?

vitamin D3 + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+

1alpha-hydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

vitamin D3 + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+

25-hydroxyvitamin D3 + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

calcidiol + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+ + O2

calcitriol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

768102, 766334, 766261

Substrates: calcidiol i.e. 25-hydroxyvitamin D3, calcitriol i.e. 1alpha,25-dihydroxyvitamin D3
Products: -

?

calciol + O2 + 2 reduced ferredoxin [iron-sulfur] cluster + 2 H+

calcidiol + 2 oxidized ferredoxin [iron-sulfur] cluster + H2O

768102, 766334, 766261

Substrates: calciol i.e. vitamin D3, calcidiol i.e. 25-hydroxyvitamin D3
Products: -

?

heme

amino acids at position 294 was located close to the heme group

766261

0.0012

1alpha,25-dihydroxyvitamin D3

26-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.0065

1alpha-hydroxyvitamin D3

25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.0022

25-hydroxyvitamin D3

1alpha-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.00091

calcidiol

25-hydroxyvitamin D3 1alpha-hydroxylation, pH 7.4, 30°C

0.00054

calciol

vitamin D3 25-hydroxylation, pH 7.4, 30°C

0.00059

vitamin D2

vitamin D2 25-hydroxylation, pH 7.4, 30°C

0.0035

vitamin D3

25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.0009

1alpha,25-dihydroxyvitamin D3

26-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.035

1alpha-hydroxyvitamin D3

25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.0023

25-hydroxyvitamin D3

1alpha-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.0023

vitamin D3

25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.72

1alpha,25-dihydroxyvitamin D3

26-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

5.5

1alpha-hydroxyvitamin D3

25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

1.03

25-hydroxyvitamin D3

1alpha-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

0.67

vitamin D3

25-hydroxylation, mutant R73V/R84A, pH not specified in the publication, temperature not specified in the publication

additional information

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737586, 737618, 737668, 737669, 738203, 766261, 766334, 768102

UniProt

Highest Expressing Human Cell Lines

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Cell Line Links

Gene Links

structure of the mutant R73V/R84A with 1alpha,25-dihydroxyvitamin D3, a hydrogen-bond network including the 1alpha-hydroxyl group and several water molecules play an important role in the substrate-binding for 26-hydroxylation

wild-type to 1.5 A resolution, and mutant R84A, native protein and in complex with 1alpha,25-dihydroxyvitamin D3. The compound is positioned 11 A from the iron atom along the I helix within the pocket. The loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B and F helices, creating a transient binding site

I243A

complete loss of activity

I293A

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 5.4 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 3.4 mmol of product per min and mol of protein

L180A

complete loss of activity

M239A

mutation generates the 1alpha-hydroxylation activity for vitamin D2

Q87W/T115A/H132L/R194W/G294D

the mutant enzyme shows a 20fold higher activity than the wild-type enzyme

766261

R193A

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 0.48 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 0.72 mmol of product per min and mol of protein

R193K

complete loss of activity

R193Q

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 0.33 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 0.31 mmol of product per min and mol of protein

R73A

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 30.9 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 20.8 mmol of product per min and mol of protein

R73A/R84A

R73A/R84A/M239A

M239A mutation induces large changes in the local conformation around Trp residues and markedly reduces the enzyme stability. M239A mutation enhances self-association

R73A/R84A/M239A/E90A

M239A mutation induces large changes in the local conformation around Trp residues and markedly reduces the enzyme stability. M239A mutation enhances self-association and E90A mutations, in cooperation with M239A, accelerates self-association with little effect on the stability

R73A/R84A/M239A/E90D

M239A mutation induces large changes in the local conformation around Trp residues and markedly reduces the enzyme stability. M239A mutation enhances self-association and E90D mutations, in cooperation with M239A, accelerates self-association with little effect on the stability

R73V/R84A

R84A

R84F

mutation gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type

737668

R89A

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 0.25 mmol of product per min and mol of protein, no 25-hydroxyvitamin D3 hydroxylation activity at C1alpha

S236A

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 4.6 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 3.4 mmol of product per min and mol of protein

V181A

complete loss of activity

V88A

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 1.52 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 0.34 mmol of product per min and mol of protein

Glycerol

strong stabilizing effect

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768102, 766334

recombinant protein

expressed in Escherichia coli BL21(DE3)

768102

expression in Escherichia coli

expression in Streptomyces lividans

industry

the production of physiologically active vitamin D3 is enhanced by engineering the hydroxylase CYP105A1 and the electron transport chain. A one-bacterium-multi-enzyme system is constructed and used in whole-cell catalytic experiments. 2.491 mg/l of 2 25-hydroxyvitamin D3 and 0.698 mg/l of 1alpha,25-dihydroxyvitamin D3 are successfully produced under the condition of 1.0% co-solvent DMSO, 1 mM coenzyme NADH and 35 g/l biocatalyst loading. The study contributes to a basis for the industrial production of active vitamin D3

768102

synthesis