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首页> 外文OA文献 >Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B▿
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Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B▿

机译：烟曲霉甾醇14-α脱甲基酶（CYP51）同工酶A和B▿的表达，纯化和鉴定

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Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (Ks, 8.6 μM) and eburicol (Ks, 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol (Ks, 3.1 μM) and eburicol (Ks, 4.1 μM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the Kd (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 μM, respectively, in comparison with a Kd value of 4 μM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with Kd values ranging from 1 μM for itraconazole to 11.9 μM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.
机译：烟曲霉固醇14-α脱甲基酶（CYP51）同工酶A（AF51A）和B（AF51B）在大肠杆菌中表达并纯化。与AF51B（CO-P450配合物稳定）相比，AF51A的连二亚硫酸盐还原的CO-P450配合物不稳定，迅速变性为无活性的P420。使用纯化的AF51B，使用羊毛甾醇（Ks，8.6μM）和eburicol（Ks，22.6μM）获得I型底物结合光谱。 AF51A的膜悬浮液同时与羊毛甾醇（Ks，3.1μM）和eburicol（Ks，4.1μM）结合。除氟康唑外，唑类与AF51B的结合紧密，克霉唑，伊曲康唑，泊沙康唑和伏立康唑的Kd（解离常数）值分别为0.21、0.06、0.12和0.42μM。氟康唑的Kd值为4μM。 AF51B获得了特征性的II型唑结合光谱，而除克霉唑以外的所有其他唑均在AF51A结合光谱中出现了一个额外的谷点和一个蓝移光谱峰。这表明AF51A的血红素修复基团内有两个不同的唑结合构象。所有五个吡咯与AF51A的结合相对较弱，Kd值范围从伊曲康唑为1μM到氟康唑为11.9μM。纯化的AF51A和AF51B的唑结合特性为烟曲霉中固有的对唑（氟康唑）的耐药性提供了解释。

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