Reconstitution and substrate specificity for isopentenyl pyrophosphate of the antiviral radical SAM enzyme viperin
Reconstitution and substrate specificity for isopentenyl pyrophosphate of the antiviral radical SAM enzyme viperin
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DOI：
10.1074/jbc.ra118.003998
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发表时间：
2018-09-07
影响因子：
4.8
通讯作者：
Huang, Raven H.
中科院分区：
文献类型：
--
作者：
Chakravarti, Arpita;Selvadurai, Kiruthika;Huang, Raven H.
Viperin 是一种自由基 SAM 酶，已被证明对多种病毒具有抗病毒活性；然而，其分子机制尚不清楚。我们在这里报告，重组真菌和古菌蝰蛇酶催化 5-脱氧腺苷基（5-dA（中心点））加成到异戊烯基焦磷酸（IPP）的双键上，产生一种新的化合物，我们将其命名为腺苷酸异戊基焦磷酸（AIPP）。该反应是 IPP 特有的，因为甲羟戊酸生物合成途径中涉及的其他焦磷酸化合物不与 5-dA（中心点）发生反应。使用 IPP 衍生物作为底物的酶反应表明，IPP 中的任何化学变化都会削弱其作为真菌蝰蛇蛋白有效底物的能力。突变研究表明，真菌蝰蛇蛋白中 Tyr-245 侧链上的羟基可能是自由基加成最后一步中的氢源，这为真菌蝰蛇蛋白催化的自由基反应提供了机制见解。基于结构的 viperin 与 IPP 相互作用的分子动力学 (MD) 模拟揭示了 IPP 的异戊烯基基序与 viperin 活性位点空腔的良好契合，揭示了 viperin 对 IPP 底物特异性的分子基础。总的来说，我们的研究结果表明，IPP 是真菌和古菌蝰蛇蛋白酶的有效底物，并为反应机制提供了重要的见解。
Viperin is a radical SAM enzyme that has been shown to possess antiviral activity against a broad spectrum of viruses; however, its molecular mechanism is unknown. We report here that recombinant fungal and archaeal viperin enzymes catalyze the addition of the 5-deoxyadenosyl radical (5-dA(center dot)) to the double bond of isopentenyl pyrophosphate (IPP), producing a new compound we named adenylated isopentyl pyrophosphate (AIPP). The reaction is specific for IPP, as other pyrophosphate compounds involved in the mevalonate biosynthetic pathway did not react with 5-dA(center dot). Enzymatic reactions employing IPP derivatives as substrates revealed that any chemical change in IPP diminishes its ability to be an effective substrate of fungal viperin. Mutational studies disclosed that the hydroxyl group on the side chain of Tyr-245 in fungal viperin is the likely source of hydrogen in the last step of the radical addition, providing mechanistic insight into the radical reaction catalyzed by fungal viperin. Structure-based molecular dynamics (MD) simulations of viperin interacting with IPP revealed a good fit of the isopentenyl motif of IPP to the active site cavity of viperin, unraveling the molecular basis of substrate specificity of viperin for IPP. Collectively, our findings indicate that IPP is an effective substrate of fungal and archaeal viperin enzymes and provide critical insights into the reaction mechanism.