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Journal of Bacteriology, February 2009, p. 1211-1219, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01276-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis: Conformational Flexibility and Histidine Ligation State{triangledown} ,{dagger}

Lian-Hua Xu,1 Shinya Fushinobu,1 Haruo Ikeda,2 Takayoshi Wakagi,1 and Hirofumi Shoun1*

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,1 Kitasato Institute for Life Sciences, Kitasato University, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan2

Received 11 September 2008/ Accepted 30 November 2008

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.

* Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone and fax: 81-3-5841-5148. E-mail: ahshoun{at}mail.ecc.u-tokyo.ac.jp

{triangledown} Published ahead of print on 12 December 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, February 2009, p. 1211-1219, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01276-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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