Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis : Conformational Flexibility and Histidine Ligation State

Abstract

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.

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

“…Protein Preparation and Spectroscopy-CYP105P1 protein was expressed and purified as described previously (29). The primers used to amplify the CYP105D6 gene were 5Ј-CCC ATA TGA CTG AGA CCG AAA TCC GCC TC-3Ј and 5Ј-GGA CTA GTT CAG TGG TGG TGG TGC CAG ACG ACG GGG AGC TCG ATC-3Ј (bold type and underlined sequences represent the restriction endonuclease sites and His 4 tag, respectively).…”

“…Spectroscopy-UV-visible absorption spectra measurements and titration experiments were performed essentially using the same methods as described previously (29). For the titrations of filipin I to CYP105P1, 1 ml of assay buffer containing 50 mM potassium phosphate (pH 7.5), 0.1 mM dithiothreitol, 0.1 mM EDTA, and 10% (v/v) glycerol was used.…”

“…Spectral characterization of purified CYP105P1 has been described previously (29). UV-visible absorption spectra of purified CYP105D6 in the ferric (resting), dithionite-reduced, and dithionite-reduced plus CO states are shown in supplemental Fig.…”

“…We previously reported the crystal structures of CYP105P1 in three different states (29). The ligand-free wild-type structure provides a unique state in which the His-72 residue in the BC loop is ligated to the heme iron atom.…”

Double electron–electron resonance shows cytochrome P450cam undergoes a conformational change in solution upon binding substrate

“…Structurally distinct substrate binding sites have been observed for bacterial (4,10,11), microsomal (12)(13)(14)(15), and mitochondrial P450s (16). In addition, several P450s have been observed to undergo significant conformational change in these structural elements upon substrate or inhibitor binding (17)(18)(19)(20)(21)(22). However, it remains unclear whether conformational change plays an important role in all P450s.…”