Crystal structures of cytochrome P450 105P1 from Streptomyces avermitilis: conformational flexibility and histidine ligation state.
The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes. The filipin biosynthetic pathway of Streptomyces avermitilis contains two position-specific hydroxylases, C26-specific CYP105P1 and C1'-specific CYP105D6. In this study, we describe the three X-ray crystal structures of CYP105P1: the ligand-free wild-type (WT-free), 4-phenylimidazole-bound wild-type (WT-4PI), and ligand-free H72A mutant (H72A-free) forms. The BC loop region in the WT-free structure has a unique feature; the side chain of His72 within this region is ligated to the heme iron. On the other hand, this region is highly disordered and widely open in WT-4PI and H72A-free structures, respectively. Histidine ligation of wild-type CYP105P1 was not detectable in solution, and a type II spectral change was clearly observed when 4-phenylimidazole was titrated. The H72A mutant showed spectroscopic characteristics that were almost identical to those of the wild-type protein. In the H72A-free structure, there is a large pocket that is of the same size as the filipin molecule. The highly flexible feature of the BC loop region of CYP105P1 may be required to accept a large hydrophobic substrate.
Xu
Lian-Hua
LH
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Fushinobu
Shinya
S
Ikeda
Haruo
H
Wakagi
Takayoshi
T
Shoun
Hirofumi
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003460:Crystallization; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005372:Filipin / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D006639:Histidine; D005372:Filipin; D003577:Cytochrome P-450 Enzyme System