論文

査読有り
2011年10月

Role of a Mutated Residue at the Entrance of the Substrate Access Channel in Cytochrome P450 Engineered for Vitamin D-3 Hydroxylation Activity

BIOCHEMISTRY
  • Hiroaki Fukunishi
  • ,
  • Hirotaka Yagi
  • ,
  • Ken'ichi Kamijo
  • ,
  • Jiro Shimada

50
39
開始ページ
8302
終了ページ
8310
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1021/bi2006493
出版者・発行元
AMER CHEMICAL SOC

The cytochrome P450 enzyme engineered for enhancement of vitamin D-3 (VD3) hydroxylation activity, Vdh-K1, includes four mutations (T70R, V156L, E216M, and E384R) compared to the wild-type enzyme. Plausible roles for V156L, E216M, and E384R have been suggested by crystal structure analysis (Protein Data Bank 3A50), but the role of T70R, which is located at the entrance of the substrate access channel, remained unclear. In this study, the role of the T70R mutation was investigated by using computational approaches. Molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed, and differences between R70 and T70 were compared in terms of structural change, binding free energy change (PMF), and interaction force between the enzyme and substrate. MD simulations revealed that R70 forms a salt bridge with D42 and the salt bridge affects the locations and the conformations of VD3 in the bound state. SMD simulations revealed that the salt bridge tends to be formed strongly when VD3 passes through the binding pocket. PMFs showed that the T70R mutation leads to energetic stabilization of enzyme-VD3 binding in the region near the heme active site. Interestingly, these results concluded that the D42-R70 salt bridge at the entrance of the substrate access channel affects the region near the heme active site where the hydroxylation of VD3 occurs; i.e., it is thought that the T70R mutation plays an important role in enhancing VD3 hydroxylation activity. A significant future challenge is to compare the hydroxylation activities of R70 and T70 directly by a quantum chemical calculation, and three-dimensional coordinates of the enzyme and VD3 obtained from MD and SMD simulations will be available for the future challenge.

Web of Science ® 被引用回数 : 12

リンク情報
DOI
https://doi.org/10.1021/bi2006493
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000295187200006&DestApp=WOS_CPL

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