α-Amine Desaturation of D-Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent L-Arginine 3-Hydroxylase, VioC
α-Amine Desaturation of D-Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent L-Arginine 3-Hydroxylase, VioC
复制标题
DOI：
10.1021/acs.biochem.8b00901
复制
发表时间：
2018-11-20
期刊：
影响因子：
2.9
通讯作者：
Boal, Amie K.
中科院分区：
文献类型：
--
作者：
Dunham, Noah P.;Mitchell, Andrew J.;Boal, Amie K.
当底物类似物挑战时，依赖铁(II)和2-(氧)戊二酸(Fe/2OG)的加氧酶可以促进不同于它们在天然底物上的转化。我们发现Fe/2OG酶，VioC，是一种天然的L-精氨酸3-羟基酶，催化其底物D-精氨酸的高效氧化脱胺。与D-Arg的反应物络合物保留了酶和底物官能团之间的所有相互作用，但所需的结构调整和C2的相反构型使该碳比C3更有利于向铁基中间体提供氢(H-中心点)。最简单的可能机理，C2羟基化，然后消除氨，与证明的产品中酮氧的溶剂来源不一致。相反，反应通过一种易水解的C2-亚胺中间体进行，证明了它在溶液中与(NaBH4)-H-2还原捕获生成外消旋[H-2]Arg。在亚胺物种的两种替代途径中，C2羟化后脱水与直接减饱和相比，后一种可能性似乎更大，因为前一种机制预计会导致O-18从O-18(2)中可检测到的掺入。这一分析所暗示的C-N键的直接脱饱和度类似于最近对L-Arg 4，5-脱饱和酶Napi反应的假设，从而支持了先前的机理提议。这样的途径也可能在Fe/2OG N-去甲基酶催化的一系列反应中发挥作用，这些酶被认为是通过甲基羟化和消除甲醛来实现C-N键的断裂。
When challenged with substrate analogues, iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases can promote transformations different from those they enact upon their native substrates. We show here that the Fe/2OG enzyme, VioC, which is natively an L-arginine 3-hydroxylase, catalyzes an efficient oxidative deamination of its substrate enantiomer, D-Arg. The reactant complex with D-Arg retains all interactions between enzyme and substrate functional groups, but the required structural adjustments and opposite configuration of C2 position this carbon more optimally than C3 to donate hydrogen (H-center dot) to the ferryl intermediate. The simplest possible mechanism, C2 hydroxylation followed by elimination of ammonia, is inconsistent with the demonstrated solvent origin of the ketone oxygen in the product. Rather, the reaction proceeds via a hydrolytically labile C2-iminium intermediate, demonstrated by its reductive trapping in solution with (NaBH4)-H-2 to produce racemic [H-2]Arg. Of two alternative pathways to the iminium species, C2 hydroxylation followed by dehydration versus direct desaturation, the latter possibility appears to be more likely, because the former mechanism would be expected to result in detectable incorporation of O-18 from O-18(2). The direct desaturation of a C-N bond implied by this analysis is analogous to that recently posited for the reaction of the L-Arg 4,5-desaturase, NapI, thus lending credence to the prior mechanistic proposal. Such a pathway could also potentially be operant in a subset of reactions catalyzed by Fe/2OG N-demethylases, which have instead been purported to enact C-N bond cleavage by methyl hydroxylation and elimination of formaldehyde.