DNA-Mediated Assembly of Cytochrome P450 BM3 Subdomains
DNA-Mediated Assembly of Cytochrome P450 BM3 Subdomains
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DOI：
10.1021/ja204993s
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发表时间：
2011-10-12
影响因子：
15
通讯作者：
Niemeyer, Christof M.
中科院分区：
文献类型：
--
作者：
Erkelenz, Michael;Kuo, Chi-Hsien;Niemeyer, Christof M.
细胞色素P450 BM3是一种用途广泛的酶，在生物催化和生物医学领域有着广阔的应用前景。我们在这里报告了基于BM3的两个亚域，还原酶域BMR和卟啉结构域BMP的DNA-蛋白质杂交装置的产生。这两个亚域都被基因融合到HaloTag蛋白上，这是一种自我标记的酶，允许与氯代烷修饰的寡核苷酸进行生物结合。亚域-DNA-嵌合体可以被互补的寡核苷酸重组，从而导致BM3全酶单加氧酶活性的重建，报告底物12-pncA的转化证明了这一点。两个嵌合体在可切换的DNA支架上的排列使人们能够控制两个亚域之间的距离，正如全酶的DNA依赖活性所表明的那样。此外，还构建了一种可切换的嵌合体装置，其中单加氧酶活性可以通过DNA链置换来关闭。这项研究表明，P450 BM3工程和DNA纳米技术的策略可以结合在一起，为开发具有潜在应用前景的新型筛选系统或响应性催化剂开辟新的途径。
Cytochrome P450 BM3 is a versatile enzyme, which holds great promise for applications in biocatalysis and biomedicine. We here report on the generation of a hybrid DNA-protein device based on the two subdomains of BM3, the reductase domain BMR and the porphyrin domain BMP. Both subdomains were fused genetically to the HaloTag protein, a self-labeling enzyme, allowing for the bioconjugation with chloroalkane-modified oligonucleotides. The subdomain-DNA-chimeras could be reassembled by complementary oligonucleotides, thus leading to reconstitution of the monooxygenase activity of BM3 holoenzyme, as demonstrated by conversion of the reporter substrate 12-pNCA. Arrangement of the two chimeras on a switchable DNA scaffold allowed one to control the distance between both subdomains, as indicated by the DNA-dependent activity of the holoenzyme. Furthermore, a switchable chimeric device was constructed, in which monooxygenase activity could be turned off by DNA strand displacement. This study demonstrates that P450 BM3 engineering and strategies of DNA nanotechnology can be merged to open up novel ways for the development of novel screening systems or responsive catalysts with potential applications in drug delivery.