近日，中科院武漢病毒研究所周寧一研究員課題組揭示了鄰硝基酚2-單加氧酶（OnpA）是一種新的含有細胞色素b5結(jié)構(gòu)域的獨特黃素單加氧酶,，細胞色素b5結(jié)構(gòu)域?qū)Υ藛渭友趺傅墓δ苁潜仨毜?。相關(guān)結(jié)果已發(fā)表在微生物學(xué)刊物Journal of Bacteriology上。
細胞色素b5是含有血紅素輔基的氧化還原蛋白,，它廣泛存在于真核生物的電子傳遞鏈中,，參與如脂肪酸去飽和反應(yīng)、細胞色素P450的還原等一系列氧化還原過程。細胞色素b5除了以單獨的蛋白參與酶的催化反應(yīng),，它還以融合蛋白的形式出現(xiàn)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賴于FAD的單加氧酶中從未見細胞色素b5結(jié)構(gòu)域以融合蛋白形式存在的報道,。
周寧一課題組在研究原核生物Alcaligenes sp. strain NyZ215降解鄰硝基酚（ONP）的代謝途徑時,，曾鑒定了鄰硝基酚2-單加氧酶OnpA催化鄰硝基酚的單加氧反應(yīng)而生成鄰苯二醌 (Xiao et al， 2007. J.Bacteriol. 189：6587-6593),。進一步對OnpA保守功能域的分析顯示,，OnpA中1-387個氨基酸的位置中存在FAD的結(jié)合結(jié)構(gòu)域，該結(jié)構(gòu)域也同樣存在于其他黃素單加氧酶中,。而在OnpA C端482至555的位置的氨基酸序列與細胞色素b5結(jié)構(gòu)域有較高的一致性,，但在其他已知功能的黃素單加氧酶中從未發(fā)現(xiàn)過這個結(jié)構(gòu)域。
通過酶學(xué)方法檢測,，OnpA蛋白被確認具有細胞色素b5氧化態(tài)以及還原態(tài)的特征光譜,，LC-MS證明其結(jié)合有血紅素。此外,，加入一定量的血紅素對于OnpA的酶活力有明顯的提高作用,。定點突變還證明，OnpA中還含有細胞色素b5的與血紅素結(jié)合的保守氨基酸,。
此研究在黃素單加氧酶中揭示了從未發(fā)現(xiàn)過的具有功能的細胞色素b5結(jié)構(gòu)域,，同時證明了原核生物中也有融合有細胞色素b5結(jié)構(gòu)域的功能蛋白的存在,。該研究揭示了原核生物中細胞色素b5與黃素單加氧酶的在功能上的關(guān)聯(lián),，對黃素蛋白以及原核生物中細胞色素b5的進一步研究具有重要的科學(xué)價值。（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.
