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Literature summary for 1.14.15.18 extracted from

  • Yasutake, Y.; Fujii, Y.; Nishioka, T.; Cheon, W.K.; Arisawa, A.; Tamura, T.
    Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase (2010), J. Biol. Chem., 285, 31193-31201.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type Vdh and mutant Vdh-K1 in Escherichia coli Pseudonocardia autotrophica

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant wild-type Vdh and mutant Vdh-K1 in complex with substrates VD3 and 25(OH)VD3, 20 mg/ml wild-type protein in 20 mM Tris-HCl, pH 7.5, supplemented with 0-0.2% PMbetaCD for VD3 complex or 0-0.2% gammaCD for 25(OH)VD3 complex, 20 mg/ml mutant protein in 20 mM Tris-HCl, pH 7.5, supplemented with 20 mM NaCl, sitting-drop method, 20°C. Vdh-WT crystals are grown using reservoir solution containing 0.1 M BisTris, pH 7.5, 50 mM CaCl2, 40-120 mM NaCl or KCl, and 32-40% PEG 400 or PEG 550 monomethyl ether or 20% PEG 1000, Vdh-K1 is crystallized using reservoir solution containing 0.1 M calcium acetate and 10-14% PEG 3350, X-ray diffraction structure determination and analysis at 1.75-3.05 A resolution Pseudonocardia autotrophica

Protein Variants

Protein Variants Comment Organism
T70R/V156L/E216M/E384R mutant Vdh-K1, 22fold more active in the hydroxylation of VD3 to 25(OH)VD3 than wild-type Vdh in in vivo bioconversion using Escherichia coli cells. backbone and side chain conformations at the active-site pocket are identical among the substrate-free, VD3-bound, and 25(OH)VD3-bound forms of Vdh-K1, and no induced-fit mechanisms are observed, although VD3 is asymmetric Pseudonocardia autotrophica

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Pseudonocardia autotrophica Vdh, a cytochrome P450 enzyme, is responsible for the biocatalytic conversion of vitamin D3, catalyzing the two-step hydroxylation of VD3, i.e. the conversion of VD3 to 25-hydroxyvitamin D3 (25(OH)VD3) and then of 25(OH)VD3 to 1alpha,25(OH)2VD3, a hormonal form of VD3. Anti-parallel substrate binding modes enable sequential hydroxylation, structure-function analysis, overview ?
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Organism

Organism UniProt Comment Textmining
Pseudonocardia autotrophica
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Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type Vdh and mutant Vdh-K1 from Escherichia coli Pseudonocardia autotrophica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information Vdh, a cytochrome P450 enzyme, is responsible for the biocatalytic conversion of vitamin D3, catalyzing the two-step hydroxylation of VD3, i.e. the conversion of VD3 to 25-hydroxyvitamin D3 (25(OH)VD3) and then of 25(OH)VD3 to 1alpha,25(OH)2VD3, a hormonal form of VD3. Anti-parallel substrate binding modes enable sequential hydroxylation, structure-function analysis, overview Pseudonocardia autotrophica ?
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Subunits

Subunits Comment Organism
More Vdh structure analysis, overview Pseudonocardia autotrophica

Synonyms

Synonyms Comment Organism
25(OH)VD3 1alpha-hydroxylase
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Pseudonocardia autotrophica
VD3 25-hydroxylase
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Pseudonocardia autotrophica
VDH
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Pseudonocardia autotrophica
vitamin D3 hydroxylase
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Pseudonocardia autotrophica