Structural Diversities of Active Site in Clinical Azole-bound Forms between Sterol 14α-Demethylases (CYP51s) from Human and Mycobacterium tuberculosis

Koji Matsuura,Shiro Yoshioka,Takehiko Tosha,Hiroshi Hori,Koichiro Ishimori,Teizo Kitagawa,Isao Morishima,Norio Kagawa,Michael R Waterman

doi:10.1074/jbc.m413042200

Abstract

To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET-bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single gx and gz values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.

Highlights

To obtain insight into molecular mechanisms for specificity of azoles, we focused on two cytochrome P450 sterol 14␣-demethylase (CYP51) from different species and characterized their azole binding properties by spectroscopies including absorption, resonance Raman, and EPR spectra
The peak at 417 nm is characteristic of the 6-coordinate low spin state (6cLS) [27, 28], indicating that a water molecule is coordinated to the heme iron
Summary—To investigate the structural origin of the inhibitor specificity in CYP51s, we characterized structural changes associated with the azole binding in bacterial and human CYP51s by electronic absorption and resonance Raman and EPR spectroscopies

Summary

EXPERIMENTAL PROCEDURES

Sample Preparation—MtCYP51 was expressed in DH5␣ and purified as described previously [23, 27,28,29,30]. We purified the enzyme by histidine-tagged column (Amersham Biosciences) with 50 mM phosphate buffer (pH 7.4) containing 20% glycerol. Expression of HuCYP51 was performed in DH5␣ as previously reported [28]. Supernatant of lysate from the cells expressing CYP51s was diluted with 50 mM Tris-HCl (pH 7.4) containing 20% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, and 20 mM mercaptoethanol and the mixture was applied onto the Ni2ϩ-nitriloacetic acid-agarose (Qiagen) column equilibrated with the same buffer. The enzyme concentration for the resonance Raman measurements was 0.05 mM. The concentrations of the inhibitors we used for the measurements were 5, 2.5, 2.5, and 5 mM for 4-PhIm, FLU, KET, and 2-PhIm, respectively. The concentrations of inhibitors for the EPR measurements were 3, 3, 3, and 10 mM for 4-PhIm, FLU, KET, and 2-PhIm, respectively

RESULTS

TABLE I

DISCUSSION