3A50

Structure of cytochrome P450 Vdh mutant (Vdh-K1) obtained by directed evolution with bound vitamin D3

Summary for 3A50

• Entry DOI: 10.2210/pdb3a50/pdb
• Related: 3A4G 3A4H 3A4Z 3A51
• Descriptor: Vitamin D hydroxylase, PROTOPORPHYRIN IX CONTAINING FE, (1S,3Z)-3-[(2E)-2-[(1R,3AR,7AS)-7A-METHYL-1-[(2R)-6-METHYLHEPTAN-2-YL]-2,3,3A,5,6,7-HEXAHYDRO-1H-INDEN-4-YLIDENE]ETHYLI DENE]-4-METHYLIDENE-CYCLOHEXAN-1-OL, ... (7 entities in total)
• Functional Keywords: cytochrome p450, vitamin d3 hydroxylase, hemoprotein, monooxygenase, directed evolution, oxidoreductase
• Biological source: Pseudonocardia autotrophica
• Cellular location: Cytoplasm : C4B644
• Total number of polymer chains: 5
• Total formula weight: 233723.98

Authors

Yasutake, Y.,Fujii, Y.,Cheon, W.K.,Arisawa, A.,Tamura, T. (deposition date: 2009-07-24, release date: 2010-07-28, Last modification date: 2023-11-01)

Primary citation

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
J.Biol.Chem., 285:31193-31201, 2010

PubMed 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 1α,25-dihydroxyvitamin D(3) (1α,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 1α,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) 1α-hydroxylase activities.

PubMed: 20667833
DOI: 10.1074/jbc.M110.147009

Experimental method

X-RAY DIFFRACTION (2.05 Å)