近日，中科院武汉病毒研究所周宁一研究员课题组揭示了邻硝基酚2-单加氧酶（OnpA）是一种新的含有细胞色素b5结构域的独特黄素单加氧酶，细胞色素b5结构域对此单加氧酶的功能是必须的。相关结果已发表在微生物学刊物Journal of Bacteriology上。
细胞色素b5是含有血红素辅基的氧化还原蛋白，它广泛存在于真核生物的电子传递链中，参与如脂肪酸去饱和反应、细胞色素P450的还原等一系列氧化还原过程。细胞色素b5除了以单独的蛋白参与酶的催化反应，它还以融合蛋白的形式出现在真核生物的sulfite oxidase，fatty acid desaturases，nitrate reductase，flavocytochrome b2等蛋白中。然而，目前在原核生物的蛋白中或在依赖于FAD的单加氧酶中从未见细胞色素b5结构域以融合蛋白形式存在的报道。
周宁一课题组在研究原核生物Alcaligenes sp. strain NyZ215降解邻硝基酚（ONP）的代谢途径时，曾鉴定了邻硝基酚2-单加氧酶OnpA催化邻硝基酚的单加氧反应而生成邻苯二醌 (Xiao et al， 2007. J.Bacteriol. 189：6587-6593)。进一步对OnpA保守功能域的分析显示，OnpA中1-387个氨基酸的位置中存在FAD的结合结构域，该结构域也同样存在于其他黄素单加氧酶中。而在OnpA C端482至555的位置的氨基酸序列与细胞色素b5结构域有较高的一致性，但在其他已知功能的黄素单加氧酶中从未发现过这个结构域。
通过酶学方法检测，OnpA蛋白被确认具有细胞色素b5氧化态以及还原态的特征光谱，LC-MS证明其结合有血红素。此外，加入一定量的血红素对于OnpA的酶活力有明显的提高作用。定点突变还证明，OnpA中还含有细胞色素b5的与血红素结合的保守氨基酸。
此研究在黄素单加氧酶中揭示了从未发现过的具有功能的细胞色素b5结构域，同时证明了原核生物中也有融合有细胞色素b5结构域的功能蛋白的存在。该研究揭示了原核生物中细胞色素b5与黄素单加氧酶的在功能上的关联，对黄素蛋白以及原核生物中细胞色素b5的进一步研究具有重要的科学价值。（生物谷Bioon.com）
doi:10.1128/JB.06411-11
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OnpA, an Unusual Flavin-Dependent Monooxygenase Containing a Cytochrome b5 Domain
Yi Xiaoa, Ting-Ting Liua, Hui Daib, Jun-Jie Zhanga, Hong Liua, Huiru Tangb, David J. Leakc and Ning-Yi Zhoua
ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b5 domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b5 domain were associated with binding of the heme, and the cytochrome b5 domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b5 domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b5 and flavin-dependent monooxygenases.
