Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2
Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2
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DOI：
10.1073/pnas.0909649106
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发表时间：
2009-10-20
影响因子：
11.1
通讯作者：
Bollinger, J. Martin, Jr.
中科院分区：
文献类型：
--
作者：
Matthews, Megan L.;Neumann, Christopher S.;Bollinger, J. Martin, Jr.
α-酮戊二酸依赖性羟化酶和卤化酶采用类似的反应机制，涉及夺氢Fe（IV）-氧代（铁酰基）中间体。在卤化酶中，来自羟化酶中发现的铁配体的His（2）（Asp/Glu）（1）“面三联体”的羧酸残基被丙氨酸取代，并且卤离子（X-）在空出的位点处配位。卤化被认为是由卤素自由基从X-Fe（III）-OH中间体“反弹”到底物自由基的结果，所述X-Fe（III）-OH中间体通过氢（H-中心点）提取产生。据报道，X-Fe（III）-OH中间体的替代衰变途径，羟基自由基反弹到底物自由基（如在羟化酶中发生的），不竞争。在这里，我们显示的卤化酶SyrB 2的烷基基团的基板远离氧代/羟基配体和更接近卤素配体的定位牺牲H-中心点提取熟练卤素反弹的选择性。在用C5氨基酸L-正缬氨酸替换L-Thr（系在天然底物中的SyrB 1载体蛋白上的C4氨基酸）后，氯铁基中间体的衰变速度加快130倍，反应结果主要转变为C5的羟基化，这与通过较长侧链将甲基投射到更靠近氧代/羟基的位置一致。竞争H-中心点提取C4的结果主要在氯化，发生在这个网站在本地基板。因此，C5的氘化，这减缓了在该位点的攻击，将区域选择性从C5切换到C4，并且将化学选择性从羟基化切换到氯化。因此，基质-中间体的配置和羧酸根->卤化物配体交换联合收割机结合以指定卤化结果。
The alpha-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His(2)(Asp/Glu)(1) "facial triad" of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X-) coordinates at the vacated site. Halogenation is thought to result from "rebound" of the halogen radical from the X-Fe(III)-OH intermediate produced by hydrogen (H-center dot) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H-center dot-abstraction proficiency for halogen-rebound selectivity. Upon replacement of L-Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid L-norvaline, decay of the chloroferryl intermediate becomes 130x faster and the reaction outcome switches to primarily hydroxylation of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H-center dot abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation to chlorination. Thus, substrate-intermediate disposition and the carboxylate -> halide ligand swap combine to specify the halogenation outcome.