Department of Chemistry, University of Adelaide, SA 5005, Australia.
School of Biological Sciences, University of Adelaide, SA 5005, Australia.
J Steroid Biochem Mol Biol. 2022 Jul;221:106097. doi: 10.1016/j.jsbmb.2022.106097. Epub 2022 Mar 25.
Members of the CYP51 family of cytochrome P450 enzymes are classified as sterol demethylases involved in the metabolic formation of cholesterol and related derivatives. The CYP51 enzyme from Mycobacterium marinum was studied and compared to its counterpart from Mycobacterium tuberculosis to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding of the purified CYP51 enzymes from M. marinum and M. tuberculosis were performed. The catalytic oxidation of lanosterol and related steroids was investigated. M. marinum CYP51 was structurally characterized by X-ray crystallography. The CYP51 enzyme of M. marinum is sequentially closely related to CYP51B1 from M. tuberculosis. However, differences in the heme spin state of each enzyme were observed upon the addition of steroids and other ligands. Both enzymes displayed different binding properties to those reported for the CYP51-Fdx fusion protein from the bacterium Methylococcus capsulatus. The enzymes were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol, but no products were detected for the related species dihydrolanosterol and eburicol. The crystal structure of CYP51 from M. marinum in the absence of added substrate but with a Bis-Tris molecule within the active site was resolved. The CYP51 enzyme of M. marinum displays differences in how steroids and other ligands bind compared to the M. tuberculosis enzyme. This was related to structural differences between the two enzymes. Overall, both of these CYP51 enzymes from mycobacterial species displayed significant differences to the CYP51 enzymes of eukaryotic species and the bacterial CYP51-Fdx enzyme of Me. capsulatus.
细胞色素 P450 酶家族 CYP51 的成员被归类为固醇脱甲基酶，参与胆固醇和相关衍生物的代谢形成。研究了来自海分枝杆菌的 CYP51 酶，并将其与结核分枝杆菌的对应物进行了比较，以确定它们之间功能保守的程度。对来自海分枝杆菌和结核分枝杆菌的纯化 CYP51 酶的底物和抑制剂结合进行了光谱分析。研究了羊毛甾醇和相关甾体的催化氧化。通过 X 射线晶体学对海分枝杆菌 CYP51 进行了结构表征。海分枝杆菌 CYP51 与结核分枝杆菌 CYP51B1 在序列上密切相关。然而，在添加类固醇和其他配体时，观察到每种酶的血红素自旋状态存在差异。两种酶对来自细菌 Methylococcus capsulatus 的 CYP51-Fdx 融合蛋白的报道的结合特性显示出不同。这些酶能够氧化脱甲基羊毛甾醇生成 14-脱甲基羊毛甾醇，但未检测到相关物种二氢羊毛甾醇和埃伯醇的产物。解析了海分枝杆菌 CYP51 在没有添加底物但在活性位点内有 Bis-Tris 分子的情况下的晶体结构。与结核分枝杆菌酶相比，海分枝杆菌 CYP51 酶在类固醇和其他配体结合方面表现出差异。这与两种酶之间的结构差异有关。总体而言，这些来自分枝杆菌物种的 CYP51 酶与真核物种的 CYP51 酶和细菌 CYP51-Fdx 酶 Me. capsulatus 表现出显著差异。
