3A4Z

Structure of cytochrome P450 Vdh mutant (Vdh-K1) obtained by directed evolution

3A4Z の概要

• エントリーDOI: 10.2210/pdb3a4z/pdb
• 関連するPDBエントリー: 3A4G 3A4H 3A50 3A51
• 分子名称: Vitamin D hydroxylase, PROTOPORPHYRIN IX CONTAINING FE, CALCIUM ION, ... (6 entities in total)
• 機能のキーワード: cytochrome p450, vitamin d3 hydroxylase, hemoprotein, monooxygenase, directed evolution, oxidoreductase
• 由来する生物種: Pseudonocardia autotrophica
• 細胞内の位置: Cytoplasm : C4B644
• タンパク質・核酸の鎖数: 5
• 化学式量合計: 231800.79

構造登録者

Yasutake, Y.,Fujii, Y.,Cheon, W.K.,Arisawa, A.,Tamura, T. (登録日: 2009-07-24, 公開日: 2010-07-28, 最終更新日: 2023-11-01)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.2 Å)