Modular behavior of tauD provides insight into the origin of specificity in α-ketoglutarate-dependent nonheme iron oxygenases
Modular behavior of tauD provides insight into the origin of specificity in α-ketoglutarate-dependent nonheme iron oxygenases
复制标题
DOI：
10.1073/pnas.0910660106
复制
发表时间：
2009-11-24
影响因子：
11.1
通讯作者：
Klinman, Judith P.
中科院分区：
文献类型：
--
作者：
McCusker, Kevin P.;Klinman, Judith P.
牛磺酸α-酮戊二酸双加氧酶（tauD）是研究最多的α-酮戊二酸（α KG）依赖性非血红素铁加氧酶之一。与所有加氧酶一样，必须在产生对所需化学反应具有足够反应性的物质和控制该物质以防止不期望的副反应之间达到良好的平衡[Klinman JP（2007）Accts Chem Res 40：325-333]。在tauD的情况下，已经显示底物氧化物质是铁酰基-氧代，并且在底物的反应性位置处引入氘导致巨大的动力学同位素效应以及氧活化与底物氧化的部分解偶联[Price JC，巴尔EW，Glass TE，Krebs C，Bollinger JM（2003）J Am Chem Soc 125：13008-13009]。我们已经在直接位于结合底物后面的位置处产生了一系列位点特异性变体（F159至L、V、A和G）。减少侧链体积减少了氧活化与C-H裂解的偶联，这通过底物氘化进一步减少。尽管有这种影响，氧活化仍然完全偶联到α KG的氧化脱羧。bis-Tris缓冲液的浓度影响氧活化与C-H裂解的偶联程度，从而暗示缓冲液参与解偶联途径。这些数据表明残基159在底物定位和tauD反应中的关键作用，并表明这些酶中的微小活性位点扰动可以允许底物反应性的变化，同时保持底物触发和氧结合/活化。
Taurine alpha-ketoglutarate dioxygenase (tauD) is one of the best-studied alpha-ketoglutarate (alpha KG)-dependent nonheme iron oxygenases. As with all oxygenases, a fine balance must be struck between generating a species sufficiently reactive for the required chemistry and controlling that species to prevent undesirable side reactions [Klinman JP (2007) Accts Chem Res 40:325-333]. In the case of tauD, the substrate oxidizing species has been shown to be a ferryl-oxo, and the introduction of deuterium at the reactive position of substrate results in an enormous kinetic isotope effect together with a partial uncoupling of oxygen activation from substrate oxidation [Price JC, Barr EW, Glass TE, Krebs C, Bollinger JM (2003) J Am Chem Soc 125: 13008-13009]. We have generated a series of site-specific variants at a position that resides directly behind bound substrate (F159 to L, V, A, and G). Decreasing side-chain bulk diminishes the coupling of oxygen activation to C-H cleavage, which is further reduced by substrate deuteration. Despite this impact, oxygen activation remains completely coupled to the oxidative decarboxylation of alpha KG. The concentration of bis-Tris buffer impacts the extent of coupling of oxygen activation to C-H cleavage, implicating the buffer in the uncoupling pathway. These data indicate a critical role for residue 159 in substrate positioning and reaction in tauD and show that minor active-site perturbations in these enzymes could allow for changes in substrate reactivity while maintaining substrate triggering and oxygen binding/activation.