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The Role of Ile476 in the Structural Stability and Substrate Binding of Human Cytochrome P450 2C8

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Abstract

The biological function and stability of a cytochrome P450 (CYP) mainly depend on the subtle properties of the residues in the active site cavity, which are generally more divergent among proteins than other parts of the protein. As the most unique member of human CYP2C family, CYP2C8 has an isoleucine (Ile) 476 instead of phenylalanine (Phe) in substrate recognizing site 6 (SRS6). However, the role of Ile476 of CYP2C8 is still unknown. Therefore, six site-directed mutants of CYP2C8 were constructed to better define this. By UV–visible and circular dichroism spectroscopy studies, we studied for the first time the structural stability and all-trans-retinoic acid binding capability of the CYP2C8 variants. We found that the ferric CYP2C8 went through three states during thermal unfolding. Combined with substrate binding studies, our data revealed that residue 476 was involved in contact with substrate and was important for maintaining the thermal stability of CYP2C8.

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Abbreviations

CYP:

Cytochrome P450

PCR:

Polymerase chain reaction

SRS:

Substrate recognizing site

CHAPS:

3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate

SDS–PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

CD:

Circular dichroism

K d :

The apparent substrate dissociation constant

ΔA max :

The extrapolated maximum spectral change

MALDI-TOF:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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Acknowledgments

We thank Prof. Eric F. Johnson (The Scripps Research Institute, California, USA) for the generous gift of the pCWOri+ plasmid, encoding cytochrome P450 2C8dH. This project was supported by the National Science Foundation of China [Approving No. 20571018].

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Authors and Affiliations

  1. Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai, 200433, China

    Lu Sun, Zhong-Hua Wang, Feng-Yun Ni, Xiang-Shi Tan & Zhong-Xian Huang

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  1. Lu Sun
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  2. Zhong-Hua Wang
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Correspondence to Xiang-Shi Tan or Zhong-Xian Huang.

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Sun, L., Wang, ZH., Ni, FY. et al. The Role of Ile476 in the Structural Stability and Substrate Binding of Human Cytochrome P450 2C8. Protein J 29, 32–43 (2010). https://doi.org/10.1007/s10930-009-9218-8

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