Department of Pharmaceutical Chemistry, University of California, San Francisco, United States.
Arch Biochem Biophys. 2011 Aug 15;512(2):197-203. doi: 10.1016/j.abb.2011.05.021. Epub 2011 Jun 17.
The essential role of human dual oxidase 2 (hDUOX2) in thyroid hormone biosynthesis defines this member of the NOX/DUOX family, whose absence due to mutation has been directly related to disease, specifically hypothyroidism. Both human DUOX isoforms, hDUOX1 and hDUOX2, are expressed in thyroid tissue; however, hDUOX1 cannot compensate for inactivation of hDUOX2, suggesting that each enzyme is differentially regulated and/or functions in a unique manner. In efforts to uncover relevant structural and functional differences we have expressed and purified the peroxidase domain of hDUOX2(1-599) for direct comparison with the previously studied hDUOX1(1-593). As was shown for hDUOX1, the truncated hDUOX2 domain purifies without a bound heme co-factor and displays no peroxidase activity. However, hDUOX2(1-599) displays greater stability than hDUOX1(1-593). Surprisingly, upon titration with heme, both isoforms bind heme with a low micromolar affinity, demonstrating that they retain a heme binding site. A conformational difference in the full-length protein and/or a protein-protein interaction may be required to increase the heme binding affinity.
人双氧化酶 2（hDUOX2）在甲状腺激素生物合成中的重要作用定义了这个 NOX/ DUOX 家族的成员，由于突变导致其缺失与疾病直接相关，特别是甲状腺功能减退症。人 DUOX 的两种同工酶，hDUOX1 和 hDUOX2，都在甲状腺组织中表达；然而，hDUOX1 不能代偿 hDUOX2 的失活，这表明每种酶的调控方式不同，或者以独特的方式发挥作用。为了揭示相关的结构和功能差异，我们表达并纯化了 hDUOX2（1-599）的过氧化物酶结构域，以便与之前研究过的 hDUOX1（1-593）进行直接比较。与 hDUOX1 一样，截短的 hDUOX2 结构域在没有结合血红素辅因子的情况下可纯化，并且没有过氧化物酶活性。然而，hDUOX2（1-599）比 hDUOX1（1-593）更稳定。令人惊讶的是，当用血红素滴定时，两种同工酶都以低微摩尔亲和力结合血红素，表明它们保留了血红素结合位点。全长蛋白的构象差异和/或蛋白质-蛋白质相互作用可能需要增加血红素的结合亲和力。
