Structural diversities of active site in clinical azole-bound forms between sterol 14alpha-demethylases (CYP51s) from human and Mycobacterium tuberculosis.
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.
Matsuura
Koji
K
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
Yoshioka
Shiro
S
Tosha
Takehiko
T
Hori
Hiroshi
H
Ishimori
Koichiro
K
Kitagawa
Teizo
T
Morishima
Isao
I
Kagawa
Norio
N
Waterman
Michael R
MR
D000595:Amino Acid Sequence; D001393:Azoles / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D058886:Sterol 14-Demethylase
D001393:Azoles; D001426:Bacterial Proteins; C548418:CYP51A1 protein, human; D011994:Recombinant Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase