Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases
Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases
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DOI：
10.1021/bi0621213
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发表时间：
2007-02-06
期刊：
影响因子：
2.9
通讯作者：
Walsh, Christopher T.
中科院分区：
文献类型：
--
作者：
Yeh, Ellen;Blasiak, Leah C.;Walsh, Christopher T.
黄素依赖卤素酶RebH催化7-氯色氨酸的形成，作为抗肿瘤药物rebeccamycin生物合成的第一步。FADH(2)、Cl-、O-2在活性位点反应生成强氧化剂HOCl，推测HOCl进行氯化反应。在此，我们证明了在没有底物色氨酸的情况下，RebH、FADH(2)、Cl-和O-2反应形成了一个长寿命的氯化中间体（t(1/2) = 63 h，温度为4℃）。该中间体在去除FAD后仍留在酶上，并以动力学合格的速率将氯转移到色氨酸。RebH的x射线晶体结构表明该中间体的身份，其活性位点Lys79位于黄素和色氨酸结合位点之间的中心位置，仅比色氨酸的C7高4.1埃。氯化产物可能是赖氨酸氯胺（lysl -epsilon NH-Cl），由酶生成的HOCl与活性位点Lys79反应而成。在这类重要的生物合成酶中，这种共价酶氯胺可能在指导底物的区域特异性氯化作用中起关键作用。
The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH(2), Cl-, and O-2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t(1/2) = 63 h at 4 degrees C) when RebH, FADH(2), Cl-, and O-2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 angstrom above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilon NH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.