Crystallization and preliminary X-ray diffraction studies of vitamin D₃hydroxylase, a novel cytochrome P450 isolated fromPseudonocardia autotrophica

Abstract

Vitamin D 3 hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D 3 via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D 3 (cholecalciferol or VD3) to 25-hydroxyvitamin D 3 [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1,25-dihydroxyvitamin D 3 [calciferol or 1,25(OH) 2 VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized. Two different crystal forms were obtained by the hanging-drop vapour-diffusion method at 293 K using polyethylene glycol as a precipitant. The form I crystal belonged to the trigonal space group P3 1 , with unit-cell parameters a = b = 61.7, c = 98.8 Å . There is one Vdh molecule in the asymmetric unit, with a solvent content of 47.6%. The form II crystal was grown in the presence of 25(OH)VD3 and belonged to the orthorhombic system P2 1 2 1 2 1 , with unit-cell parameters a = 63.4, b = 65.6 c = 102.2 Å . There is one Vdh molecule in the asymmetric unit, with a solvent content of 46.7%. Native data sets were collected to resolutions of 1.75 and 3.05 Å for form I and form II crystals, respectively, using synchrotron radiation. The structure solution was obtained by the molecular-replacement method and model refinement is in progress for the form I crystal.

Structural Evidence for Enhancement of Sequential Vitamin D3 Hydroxylation Activities by Directed Evolution of Cytochrome P450 Vitamin D3 Hydroxylase

“…Overproduction of Vdh-WT was carried out using a Rhodococcus erythropolis expression system (19) or E. coli, and purified by Ni-affinity chromatography and anion exchange chromatography, as previously described (10,20). The highly active Vdh-K1 mutant (T70R/V156L/E216M/ E384R) was generated by directed evolution (10).…”

The Activating Enzymes of Vitamin D Metabolism (25- and 1α-Hydroxylases)

“…Even with such data, the CYP2R1 and CYP105A1 structures would not reveal mechanistically how the hydrophobic parts of vitamin D substrates interact with the predominantly hydrophobic residues lining the active site to position the hydroxylation target carbon so precisely for reaction. Hopefully, a clearer picture of substrate binding will be forthcoming in substrate-bound crystal structures of the mammalian vitamin D hydroxylases or the pending release of P450 Vdh mutants obtained by directed evolution co-crystallized with vitamin D 3 (3a50.pdb) and 25(OH)D 3 (3a51.pdb) [115]. In the meantime, continuing studies of vitamin D docking in CYP27A1, CYP27B1, and CYP24A1 will focus on naturally occurring autosomal mutations [e.g., 117] (Fig.…”

“…In the last 2 years, the first crystal structures of vitamin-D-related CYPs in the form of the human microsomal CYP2R1 and the rat mitochondrial CYP24A1 have been released [18,19] and these have validated the homology modeling approach very well. In addition, two bacterial vitamin D hydroxylases capable of sequentially hydroxylating vitamin D 3 to 1a,25 (OH) 2 D 3 at production levels, CYP105A1 from Streptomyces griseolus (2zbz.pdb) [114] and P450 Vdh from Pseudonocardia autotrophica (3a4g.pdb) [115], have been determined.…”

Cytochrome P450-mediated metabolism of vitamin D

“…In addition, two bacterial vitamin D hydroxylases capable of sequentially hydroxylating vitamin D 3 to 1,25-(OH) 2 D 3 at production levels, CYP105A1 from Streptomyces griseolus (2zbz.pdb) ( 25 ) and P450 Vdh from Pseudonocardia autotrophica (3a4g. pdb) ( 26 ), have been determined. Mutational analyses to pinpoint amino-acid residues involved in contact with the main functional groups (hydroxyls) or hydrophobic cis -triene of the vitamin D substrate have been largely completed.…”