Characterization and catalytic properties of the sterol 14α-demethylase from Mycobacterium tuberculosis
Characterization and catalytic properties of the sterol 14α-demethylase from Mycobacterium tuberculosis
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DOI：
10.1073/pnas.96.16.8937
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发表时间：
1999-08-03
影响因子：
11.1
通讯作者：
Waterman, MR
中科院分区：
文献类型：
--
作者：
Bellamine, A;Mangla, AT;Waterman, MR
甾醇14 α-脱甲基酶是由CYP 51编码的一种参与真核生物甾醇合成的多功能氧化酶。结核分枝杆菌基因组计划的完成揭示了与哺乳动物14 α-脱甲基酶具有同源性的蛋白质可能存在于该细菌中。使用基因组DNA。从分枝杆菌菌株H（37）Rv，我们已经明确地确定CYP 51样基因编码细菌甾醇14 α-脱甲基酶。M.结核病CYP 51基因在大肠杆菌中产生P450，当纯化至均一性时，其具有预测的分子量，约为100。50 kDa的SDS/PAGE上，并结合甾醇底物和P450 14 α-脱甲基酶的唑类抑制剂。它催化羊毛甾醇、24，25-二氢羊毛甾醇和钝叶醇的14 α-去甲基化以立体选择性地产生8，14-二烯，如GC/MS和H-1 NMR分析所示。黄素氧还蛋白和铁氧还蛋白氧化还原系统都能够支持这种酶活性。14 α-甲基和δ（8（9））-键的结构要求通过比较在单一分子特征上不同的甾醇底物对的结合来建立，例如，环阿屯烯醇与羊毛甾醇配对。这些底物要求类似于对植物和动物P450 14 α-脱甲基酶建立的那些。从组合的结果，底物的活性位点内的官能团的相互关系表明，氧化部分的甾醇生物合成途径中存在于原核生物。
Sterol 14 alpha-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14 alpha-demethylases might be present in this bacterium. Using genomic DNA. from mycobacterial strain H(37)Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14 alpha-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14 alpha-demethylases. It catalyzes 14 alpha-demethylation of lanosterol, 24,25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and H-1 NMR analysis, Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14 alpha-methyl group and Delta(8(9))-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14 alpha-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.