title: The role of Ile476 in the structural stability and substrate binding of human cytochrome P450 2C8.
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.
authors.affiliation: Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai, 200433, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Sun
authors.forename: Zhong-Hua
authors.initials: ZH
authors.lastname: Wang
authors.forename: Feng-Yun
authors.initials: FY
authors.lastname: Ni
authors.forename: Xiang-Shi
authors.initials: XS
authors.lastname: Tan
authors.forename: Zhong-Xian
authors.initials: ZX
authors.lastname: Huang
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D065727:Cytochrome P-450 CYP2C8; D006801:Humans; D007532:Isoleucine / Q000737:chemistry*; D008968:Molecular Conformation; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D007532:Isoleucine; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8