Matthews Megan L, Neumann Christopher S, Miles Linde A, Grove Tyler L, Booker Squire J, Krebs Carsten, Walsh Christopher T, Bollinger J Martin
Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17723-8. doi: 10.1073/pnas.0909649106. Epub 2009 Oct 6.
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()) 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()-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(*) 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.
α-酮戊二酸依赖性羟化酶和卤化酶采用相似的反应机制，涉及夺取氢的Fe(IV)-氧（铁酰基）中间体。在卤化酶中，羟化酶中发现的铁配体His(2)(Asp/Glu)(1)“面三联体”中的羧酸盐残基被丙氨酸取代，并且卤离子(X⁻)在空出的位点配位。卤化被认为是由氢(H*)夺取产生的X-Fe(III)-OH中间体的卤素自由基“回弹”到底物自由基所致。据报道，X-Fe(III)-OH中间体的另一种衰变途径，即羟基自由基回弹到底物自由基（如羟化酶中发生的那样）并不具有竞争性。在此，我们证明对于卤化酶SyrB2，底物的烷基远离氧/羟基配体并更靠近卤素配体的定位牺牲了H夺取能力以换取卤素回弹选择性。用C5氨基酸L-正缬氨酸取代天然底物中与SyrB1载体蛋白相连的C-4氨基酸L-苏氨酸后，氯铁酰中间体的衰变速度加快130倍，反应结果转变为主要是C5的羟基化，这与较长侧链使甲基更靠近氧/羟基一致。从C4竞争性夺取H主要导致氯化，就像天然底物中该位点发生的那样。因此，C5的氘代减缓了该位点的攻击，既将区域选择性从C5转变为C4，又将化学选择性从羟基化转变为氯化。因此，底物-中间体的布局和羧酸盐→卤化物配体交换共同决定了卤化结果。
