Crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase, a novel cytochrome P450 isolated from Pseudonocardia 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 1alpha,25-dihydroxyvitamin D(3) [calciferol or 1alpha,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 A. 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 A. 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 A 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.

Figure 1

Crystals of Vdh. (a) The initial crystals were obtained in 100 mM bis-tris pH 6.5, 30%(w/v) PEG 550 MME and 50 mM CaCl₂. (b) After optimization of the crystallization conditions, a large hexagonal rod-shaped crystal was obtained in 100 mM bis-tris pH 7.5, 38%(w/v) PEG 400, 50 mM CaCl₂ and 60 mM NaCl. (c) Needle-shaped crystals were also obtained in the presence of 25(OH)VD3 in 100 mM bis-tris pH 7.5, 26%(w/v) PEG 1000 and 50 mM CaCl₂.

Figure 2

Examples of the X-ray diffraction images that were obtained by exposing the form I crystal at different positions. Good (left) and poor (right) diffraction images were obtained when the edge and midpoint of the same crystal were exposed to X-­rays, respectively.