3A4H: Structure Of Cytochrome P450 Vdh From Pseudonocardia Autotrophica (Orthorhombic Crystal Form)

Citation:
Abstract
Vitamin D(3) hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D(3) (VD(3)) to 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)VD(3)) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD(3), i.e. the conversion of VD(3) to 25-hydroxyvitamin D(3) (25(OH)VD(3)) and then of 25(OH)VD(3) to 1alpha,25(OH)(2)VD(3), a hormonal form of VD(3). Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD(3)-bound, and 25(OH)VD(3)-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substrate-bound structure of Vdh-K1 accommodates both VD(3) and 25(OH)VD(3) but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD(3) hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD(3) 25-hydroxylase and 25(OH)VD(3) 1alpha-hydroxylase activities.
PDB ID: 3A4HDownload
MMDB ID: 83287
PDB Deposition Date: 2009/7/7
Updated in MMDB: 2010/11
Experimental Method:
x-ray diffraction
Resolution: 3.06  Å
Source Organism:
Similar Structures:
Biological Unit for 3A4H: monomeric; determined by author and by software (PISA)
Molecular Components in 3A4H
Label Count Molecule
Protein (1 molecule)
1
Vitamin D Hydroxylase
Molecule annotation
Chemicals (2 molecules)
1
1
2
1
* Click molecule labels to explore molecular sequence information.

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