Metal ligand substitution and evidence for quinone formation in taurine/α-ketoglutarate dioxygenase
Metal ligand substitution and evidence for quinone formation in taurine/α-ketoglutarate dioxygenase
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DOI：
10.1016/j.jinorgbio.2007.01.011
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发表时间：
2007-05-01
影响因子：
3.9
通讯作者：
Hausinger, Robert P.
中科院分区：
文献类型：
--
作者：
Grzyska, Piotr K.;Muller, Tina A.;Hausinger, Robert P.
对原型 Fe-II/α-酮戊二酸 (α KG) 依赖性羟化酶、牛磺酸/α KG 双加氧酶 (TauD) 的三个金属结合配体进行系统突变，以检查各种配体取代对酶活性和金属中心特性的影响。 His99 与 alpha KG 和 Fell 共面，在维持酶活性方面是不可改变的。 Asp101 只能被更长的羧酸盐取代，D101E 变体的 kat 是野生型酶的 22%，K-cat 是野生型酶的三倍。 His255 位于 O-2 结合位点对面，对活性不太重要，可以被 Gln 甚至带负电的 Glu 取代（活性分别为 81% 和 33%）。对三种高活性突变蛋白的瞬时动力学研究揭示了野生型酶中报道的假定的 Fe-IV-oxo 中间体，但具有不同的动力学。在缓冲液中补充甲酸盐可增强 D101A 变体的活性，这与部分化学拯救缺失金属配体的结果一致。结合 Fe-II 后，野生型 TauD 和三种高活性变体的厌氧样品会产生类似于儿茶酚 Fe(II) 物质的弱绿色发色团。有证据表明，二羟基苯丙氨酸的醌氧化态是由需氧细菌生长过程中 TauD 蛋白质侧链异常自羟基化形成的，与 Fe-II 反应形成该物种。在 α KG 和牛磺酸存在的情况下，检查了与 Fe-II-TauD 和 Co-II-TauD 相关的光谱的所有变体，以获得对影响金属中心的扰动的更多见解。这些研究首次对 Fe-II/α KG 依赖性羟化酶中的金属中心配体进行系统突变分析。 (c) 2007 Elsevier Inc. 保留所有权利。
The three metal-binding ligands of the archetype Fe-II/alpha-ketoglutarate (alpha KG)-dependent hydroxylase, taurine/alpha KG dioxygenase (TauD), were systematically mutated to examine the effects of various ligand substitutions on enzyme activity and metallocenter properties. His99, coplanar with alpha KG and Fell, is unalterable in terms of maintaining an active enzyme. Asp101 can be substituted only by a longer carboxylate, with the D101E variant exhibiting 22% the kat and threefold the K-cat of wild-type enzyme. His255, located opposite the O-2-binding site, is less critical for activity and can be substituted by Gln or even the negatively charged Glu (81% and 33% active, respectively). Transient kinetic studies of the three highly active mutant proteins reveal putative Fe-IV-oxo intermediates as reported in wild-type enzyme, but with distinct kinetics. Supplementation of the buffer with formate enhances activity of the D101A variant, consistent with partial chemical rescue of the missing metal ligand. Upon binding Fe-II, anaerobic samples of wild-type TauD and the three highly active variants generate a weak green chromophore resembling a catecholate-Fe(II)species. Evidence is presented that the quinone oxidation state of dihydroxyphenylalanine, formed by aberrant self-hydroxylation of a protein side chain of TauD during aerobic bacterial growth, reacts with Fe-II to form this species. The spectra associated with Fe-II-TauD and Co-II-TauD in the presence of alpha KG and taurine were examined for all variants to gain additional insights into perturbations affecting the metallocenter. These studies present the first systematic mutational analysis of metallocenter ligands in an Fe-II/alpha KG-dependent hydroxylase. (c) 2007 Elsevier Inc. All rights reserved.