New insights into the mechanism of enzymatic chlorination of tryptophan.
New insights into the mechanism of enzymatic chlorination of tryptophan.
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DOI：
10.1002/anie.200802466
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发表时间：
2008
影响因子：
16.6
通讯作者：
van Pee, Karl-Heinz
中科院分区：
文献类型：
--
作者：
Flecks, Silvana;Patallo, Eugenio R.;Zhu, Xiaofeng;Ernyei, Aliz J.;Seifert, Gotthard;Schneider, Alexander;Dong, Changjiang;Naismith, Janies H.;van Pee, Karl-Heinz
黄素依赖性卤化酶在生物卤化反应中起重要作用。对于卤化活性，黄素依赖性卤化酶需要还原的FAD，其由FAD和NADH通过第二种酶黄素还原酶形成。虽然在许多情况下，黄素还原酶基因存在于卤代代谢酶的生物合成基因簇中，但尚不清楚相应的黄素还原酶是否直接与卤代酶相互作用。至少在许多情况下，来自不同细菌菌株的黄素还原酶可以与卤化酶组合使用。1，2-5对于来自荧光假单胞菌BL 915的色氨酸7-卤化酶PrnA，其催化硝吡咯生物合成的第一步6，可以表明，在卤化反应中，甚至化学还原的FAD也被卤化酶使用。7基于PrnA的三维结构，推测黄素氢过氧化物是由卤素酶结合的还原黄素与氧反应形成的。这种黄素氢过氧化物然后与氯离子反应，导致形成次氯酸，然后引导次氯酸沿着沿着约10 μ m长的通道朝向底物色氨酸（图1）。赖氨酸残基（K79）被认为与次氯酸形成氢键，从而使其与色氨酸反应。8 Yeh等人证明了通过HOCl与赖氨酸的ε-氨基反应形成氯胺，9表明活性剂是氯胺而不是HOCl。K79的重要性是无可争议的; K79与丙氨酸残基的交换导致卤化活性的完全丧失，如PrnA 8和来自瑞贝卡霉素生物合成的色氨酸7-卤化酶RebH所证明的。9然而，其他因素也一定在起作用，因为色氨酸的氯化不能通过溶液中的氯胺（或HOCl）完成。10，11氯胺是比HOCl弱的卤化剂，12并且根据量子力学计算，N-氯胺的形成降低了氯物质的亲电性;换句话说，与游离HOCl中的Q（Cl）=+0.017相比，电荷Q（Cl）降低至-0.07。在活性位点中，谷氨酸346（E346）位于K79的隧道对面，底物色氨酸的定位由吲哚环的NH基团与E346和丝氨酸347（S347）之间的肽键氧之间的氢键支持（图1）。E346可能参与催化循环的证据是观察到E346 Q的活性低两个数量级。[8]尽管K79在迄今已知的所有黄素依赖性卤化酶中是绝对保守的，但E346和S347仅在黄素依赖性色氨酸卤化酶中是保守的，并且它们不存在于作用于具有苯酚或吡咯环的底物的卤化酶中。Dong等人认为，E346是必需的。
Flavin-dependent halogenases have been shown to play a major role in biological halogenation reactions. For halogenating activity, flavin-dependent halogenases require reduced FAD, which is formed from FAD and NADH by a second enzyme, a flavin reductase. Although in a number of cases, a flavin reductase gene is present in the biosynthetic gene cluster of the halometabolites, it is unclear whether the corresponding flavin reductases interact directly with the halogenases. At least in a number of cases, flavin reductases from different bacterial strains can be used in combination with halogenases. 1, 2–5 For the tryptophan 7-halogenase PrnA from Pseudomonas fluorescens BL915 which catalyzes the first step in pyrrolnitrin biosynthesis6 it could be shown that even chemically reduced FAD is used by the halogenase in the halogenation reaction. 7 Based on the threedimensional structure of PrnA, it was postulated that flavin hydroperoxide is formed by the reaction of halogenase-bound reduced flavin with oxygen. This flavin hydroperoxide then reacts with chloride ion leading to the formation of hypochlorous acid, which is then guided along a tunnel about 10 Å long towards the substrate tryptophan (Figure 1). A lysine residue (K79) was suggested to hydrogen bond with hypochlorous acid and thus position it to react with tryptophan. 8 Yeh et al. demonstrated chloramine formation by the reaction of HOCl with the ε-amino group of lysine, 9 suggesting that chloramine rather than HOCl is the active agent. The importance of K79 is undisputed; exchange of K79 against an alanine residue leads to total loss of halogenating activity as demonstrated for PrnA8 and for the tryptophan 7-halogenase RebH from rebeccamycin biosynthesis. 9 However, other factors must also be at work, since chlorination of tryptophan cannot be accomplished by chloramine (or HOCl) in solution. 10, 11 Chloramine is a weaker halogenating agent than HOCl, 12 and according to quantum mechanical calculations, N-chloramine formation reduces the electrophilicity ofthe chlorine species; in other words, the charge Q (Cl) is reduced to− 0.07 compared to Q (Cl)=+ 0.017 in free HOCl.In the active site, glutamate 346 (E346) is positioned across the tunnel from K79, and the positioning of the substrate tryptophan is supported by a hydrogen bond between the NH group of the indole ring and the peptide bond oxygen between E346 and serine 347 (S347)(Figure 1). Evidence that E346 could be involved in the catalytic cycle was the observation that E346Q is two orders of magnitude less active. 8 Whereas K79 is absolutely conserved in all the flavin-dependent halogenases known so far, E346 and S347 are conserved only in flavin-dependent tryptophan halogenases and they are not present in halogenases acting on substrates with a phenol or pyrrole ring. Dong et al. suggested that E346 is required for the
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色氨酸7-卤代酶（PRNA）结构提出了一种区域选择性氯化的机制。
DOI：
10.1126/science.1116510
发表时间：
2005-09-30
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Dong C;Flecks S;Unversucht S;Haupt C;van Pée KH;Naismith JH
通讯作者：
Naismith JH
DOI：
10.1073/pnas.0506964102
发表时间：
2005-09-27
影响因子：
11.1
作者：
Dorrestein, PC;Yeh, E;Walsh, CT
通讯作者：
Walsh, CT
影响因子：
1.8
作者：
Seibold, Corina;Schnerr, Helge;van Pee, Karl-Heinz
通讯作者：
van Pee, Karl-Heinz
影响因子：
3.2
作者：
Nowak-Thompson, B;Chaney, N;Loper, JE
通讯作者：
Loper, JE
影响因子：
2.9
作者：
Yeh, Ellen;Blasiak, Leah C.;Walsh, Christopher T.
通讯作者：
Walsh, Christopher T.