Roy Subhendu, Kästner Johannes
Institute for Theoretical Chemistry, University of Stuttgart, Pfaffenwaldring 55, 70569, Stuttgart, Germany.
Chemistry. 2017 Jul 3;23(37):8949-8962. doi: 10.1002/chem.201701286. Epub 2017 Jun 14.
Salicylate 1,2-dioxygenase (SDO) was the first enzyme discovered, in the family of iron dioxygenases, to catalyze the ring cleavage of a monohydroxylated aromatic compound, salicylate, without a proton donor. Salicylate is not electron-rich like the familiar dihydroxy or aromatic substrates with an electron-donating group that are utilized in the well-known dioxygenases. SDO carries out the intramolecular C-C bond cleavage in salicylate bearing the OH and COOH groups with high regioselectivity in comparison with the extradiol and intradiol dioxygenases. The catalytic cleavage of a nonactivated substrate like salicylate that lacks an electron-donating group, also in the absence of a proton source, raises many puzzling questions about the oxy intermediates in the reaction pathway of dioxygenase enzymes in general. To answer these fundamental queries, we have investigated the full catalytic mechanism of SDO by a combination of quantum mechanics and molecular mechanics (QM/MM) calculations. Herein, our QM/MM study has several unexpected and interesting implications for the mechanistic pathway of SDO in comparison to the experimental observations. Importantly, it unravels the basis for the unexpected "intra"-cleavage regioselectivity in SDO. Ostensibly a similar alkylperoxo intermediate is formed in SDO much like in the extradiol and intradiol dioxygenases. In stark contrast to the two diol enzymes, the O-O bond breaking leads to an unprotonated gem-hydroxy carboxylate intermediate, a paradigm analogue of the elusive gem diol intermediate. This unprotonated gem-hydroxy carboxylate intermediate exclusively dictates the C-C cleavage regiospecificity in SDO, which is unprecedented in the family of dioxygenases. It forms a seven-membered lactone species, which eventually forms the ring-cleavage final product by incorporation of two oxygen atoms in the salicylate. Thus, our computational study unravels a detailed reaction pathway of the oxidative cleavage of salicylate without a proton source by identifying the hitherto elusive intermediates in the catalytic cycle of SDO with testable predictions. Moreover, we describe the atomistic origin of the new catalytic role of Arg127 in the catalysis and regioselectivity of SDO. It also rationalizes the formation of a side product without invoking a dioxetane intermediate. This study is important from a fundamental perspective and opens up a new window in the mechanism of the family of dioxygenase enzymes.
水杨酸1,2-双加氧酶（SDO）是在铁双加氧酶家族中发现的第一种酶，它能在没有质子供体的情况下催化单羟基化芳香化合物水杨酸的环裂解。水杨酸不像那些在著名的双加氧酶中被利用的带有供电子基团的常见二羟基或芳香底物那样富电子。与二醇外切酶和二醇内切酶相比，SDO能以高区域选择性进行带有羟基和羧基的水杨酸分子内碳-碳键裂解。像水杨酸这样缺乏供电子基团的非活化底物在没有质子源的情况下的催化裂解，总体上引发了许多关于双加氧酶反应途径中氧中间体的令人困惑的问题。为了回答这些基本问题，我们通过量子力学和分子力学（QM/MM）计算相结合的方法研究了SDO的完整催化机制。在此，与实验观察结果相比，我们的QM/MM研究对SDO的反应机理途径有几个意想不到且有趣的启示。重要的是，它揭示了SDO中意想不到的“分子内”裂解区域选择性的基础。表面上，SDO中形成了一个与二醇外切酶和二醇内切酶中类似的烷基过氧中间体。与这两种二醇酶形成鲜明对比的是，O - O键断裂产生了一个未质子化的偕羟基羧酸盐中间体，这是难以捉摸的偕二醇中间体的一个范例类似物。这个未质子化的偕羟基羧酸盐中间体唯一地决定了SDO中的碳-碳裂解区域特异性，这在双加氧酶家族中是前所未有的。它形成了一种七元内酯物种，最终通过在水杨酸中引入两个氧原子形成环裂解最终产物。因此，我们的计算研究通过确定SDO催化循环中迄今难以捉摸的中间体并做出可测试的预测，揭示了在没有质子源的情况下水杨酸氧化裂解的详细反应途径。此外，我们描述了Arg127在SDO催化和区域选择性中的新催化作用的原子起源。它还解释了在不涉及二氧杂环丁烷中间体的情况下副产物的形成。这项研究从基础角度来看很重要，并为双加氧酶家族的机制打开了一扇新窗口。
