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Article

Crystal Structure of CYP105A1 (P450SU-1) in Complex with 1α,25-Dihydroxyvitamin D₃^†^,^‡

Supporting Info

Hiroshi Sugimoto*^§, Raku Shinkyo, Keiko Hayashi, Sachiyo Yoneda, Masato Yamada, Masaki Kamakura, Shin-ichi Ikushiro, Yoshitsugu Shiro^§ and Toshiyuki Sakaki*

RIKEN SPring-8 Center, Harima Institute, Sayo, Hyogo 679-5148, Japan, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, and Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan

Biochemistry, 2008, 47 (13), pp 4017–4027

DOI: 10.1021/bi7023767

Publication Date (Web): March 4, 2008

†

This work was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology grant (to Y.S., H.S. and T.S.) and the Sankyo Foundation of Life Sciences (to T.S.).

‡

The atomic coordinates and structure factors (PDB codes 2ZBX, 2ZBY, and 2ZBZ) have been deposited in the Protein Data Bank (PDB), Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* To whom correspondence should be addressed. Telephone: +81-791-58-2817 . Fax: +81-791-58-2818. E-mail: sugimoto@spring8.or.jp (H.S.); Telephone: +81-766-56-7500 . Fax: +81-766-56-2498. E-mail: tsakaki@pu-toyama.ac.jp (T.S.).

RIKEN SPring-8 Center.

Kyoto University.

Toyama Prefectural University.

Abstract

Vitamin D₃ (VD₃), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD₃ requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD₃ into 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1α,25(OH)₂D₃. The compound 1α,25(OH)₂D₃ is positioned 11 Å from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1−VD₃ complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B′ and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1α-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD₃.