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

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

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?

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

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737618

reaction of mutants R73A/R84A and R73V/R84A

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?

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

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

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737618

reaction of mutants R73A/R84A and R73V/R84A

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?

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

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?

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

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

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?

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

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

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737586, 737618

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?

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

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

UniProt

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

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

variant is also capable of converting vitamin D2 to its active form, that is, 1alpha,25-dihydroxyvitamin D2, via 25-hydroxyvitamin D2, with its 1alpha-hydroxylation activity toward 25-hydroxyvitamin D2 being much lower than that toward 25-hydroxyvitamin D3. The substitutions enhance both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25-hydroxyvitamin D2 from the substrate-binding pocket

737618

R73V/R84A

R84A

1alpha-hydroxyvitamin D3 hydroxylation activity at C25 75.7 mmol of product per min and mol of protein, 25-hydroxyvitamin D3 hydroxylation activity at C1alpha 34.5 mmol of product per min and mol of protein

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

recombinant protein

expression in Escherichia coli

expression in Streptomyces lividans

synthesis

expression of mutant R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter leads to synthesis of 25-hydroxyvitamin D3 and large amounts of 1alpha,25-dihydroxyvitamin D3. In addition, polar metabolites 1alpha,25(R),26-trihydroxyvitamin D3 and 1alpha,25(R),26-trihydroxyvitamin D3 are observed at a ratio of 5:1