Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine
Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine
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DOI：
10.1021/acs.biochem.9b00006
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发表时间：
2019-04-30
期刊：
影响因子：
2.9
通讯作者：
Liu, Aimin
中科院分区：
文献类型：
--
作者：
Li, Jiasong;Koto, Teruaki;Liu, Aimin
半胱氨酸双加氧酶（CDO）是一种非血红素铁酶，它将双氧中的两个氧原子加到L-半胱氨酸的硫原子上。在哺乳动物CDO的铁位点附近，有一个后消化产生的Cys-Tyr辅因子，其存在显著增强加氧酶活性。CDO中Cys-Tyr辅因子的形成是一个自催化过程，并且通过传统技术进行研究是具有挑战性的，因为交联反应是隐藏在L-半胱氨酸氧化的多个周转中的侧、非偶联、单周转氧化。在这里，我们利用最近的成功，通过遗传密码扩展策略在交联位点特异性掺入3，5-二氟-L-酪氨酸（F-2-Tyr），获得了纯粹非交联的人CDO。使用EPR光谱，我们表明，一氧化氮（（NO）-N-中心点），氧的替代品，类似地结合到未交联的F-2-Tyr 157 CDO在野生型人类CDO。我们测定了未交联的F-2-Tyr 157 CDO和成熟的野生型CDO与L-半胱氨酸和（NO）-N-中心点的复合物的X射线晶体结构。这些结构数据揭示了活性位点半胱氨酸（人酶中的Cys 93），而不是通常预期的酪氨酸（即，Tyr 157），如果正常的氧化反应解偶联，则被很好地排列以被氧化。这种基于结构的理解是进一步支持的计算研究与模型建立在未交联的三元复合物结构。总之，这些结果强烈表明，在铁辅助的Cys-Tyr辅因子生物发生过程中氧化的第一个靶点是Cys 93。基于这些数据，提出了一个合理的反应机制，实施半胱氨酸自由基参与交联形成。
Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of L-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the cross-linking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of L-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-L-tyrosine (F-2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide ((NO)-N-center dot), an oxygen surrogate, similarly binds to uncross-linked F-2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F-2-Tyr157 CDO and mature wild-type CDO in complex with both L-cysteine and (NO)-N-center dot. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidation reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the cross-link formation is proposed.