Orchestration of enzymatic processing by thiazole/oxazole-modified microcin dehydrogenases.
Orchestration of enzymatic processing by thiazole/oxazole-modified microcin dehydrogenases.
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DOI：
10.1021/bi401529y
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发表时间：
2014-01-21
期刊：
影响因子：
2.9
通讯作者：
Mitchell, Douglas A.
中科院分区：
文献类型：
--
作者：
Melby, Joel O.;Li, Xiangpo;Mitchell, Douglas A.
噻唑/恶唑修饰的微菌素（TOP13）包括结构多样的天然产物家族，其具有通过后取代安装的噻唑（in）e和恶唑（in）e杂环的存在而连接的不同的生物活性。对来自芽孢杆菌属Al Hakam（Balh）的TOMM生物合成酶的详细研究为杂环生物合成提供了重要的见解。噻唑和恶唑通过ATP依赖性环己基转移酶（C-和D-蛋白）和FMN依赖性脱氢酶（B-蛋白）的连续作用而安装，它们分别负责唑啉形成和唑啉氧化。虽然有几项研究集中在唑啉形成的机制，但关于脱氢酶（B蛋白）在整个底物加工中的作用的许多细节仍然未知。在这项工作中，我们评估了参与的脱氢酶在确定环形成的顺序，以及滥交的Balh和microcin B17 cycloproteinatases接受一个面板的noncognate cycloproteinases。为了支持所观察到的混杂性，使用荧光偏振测定法，使用FMN辅因子的固有荧光来测量脱氢酶与环己基琥珀酸酶的结合。最后，非同源酶显示出具有不依赖于环己基琥珀酸酶的活性。先前的研究鉴定了保守的Lys-Tyr基序对脱氢酶活性是重要的。使用本研究中开发的工具，显示Lys-Tyr基序不会改变与环己基转移酶的复合物形成，也不会改变还原电位。采取与已知的晶体结构的同系物，我们的数据表明，赖氨酸-酪氨酸基序是催化的重要性。总的来说，这项研究提供了一个更高层次的洞察TOMM生物合成过程中酶活性的复杂编排。
Thiazole/oxazole-modified microcins (TOMMs) comprise a structurally diverse family of natural products with varied bioactivities linked by the presence of posttranslationally installed thiazol(in)e and oxazol(in)e heterocycles. The detailed investigation of the TOMM biosynthetic enzymes from Bacillus sp. Al Hakam (Balh) has provided significant insight into heterocycle biosynthesis. Thiazoles and oxazoles are installed by the successive action of an ATP-dependent cyclodehydratase (C- and D-protein) and a FMN-dependent dehydrogenase (B-protein), which are responsible for azoline formation and azoline oxidation, respectively. Although several studies have focused on the mechanism of azoline formation, many details regarding the role of the dehydrogenase (B-protein) in overall substrate processing remain unknown. In this work, we evaluated the involvement of the dehydrogenase in determining the order of ring formation, as well as the promiscuity of the Balh and microcin B17 cyclodehydratases to accept a panel of noncognate dehydrogenases. In support of the observed promiscuity, a fluorescence polarization assay was utilized to measure binding of the dehydrogenase to the cyclodehydratase using the intrinsic fluorescence of the FMN cofactor. Ultimately, the noncognate dehydrogenases were shown to possess cyclodehydratase-independent activity. A previous study identified a conserved Lys-Tyr motif to be important for dehydrogenase activity. Using the tools developed in this study, the Lys-Tyr motif was shown to not alter complex formation with the cyclodehydratase nor the reduction potential. Taken with the known crystal structure of a homolog, our data suggest that the Lys-Tyr motif is of catalytic importance. Overall, this study provides a greater level of insight into the complex orchestration of enzymatic activity during TOMM biosynthesis.
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影响因子：
16.8
作者：
Koehnke, Jesko;Bent, Andrew;Houssen, Wael E.;Zollman, David;Morawitz, Falk;Shirran, Sally;Vendome, Jeremie;Nneoyiegbe, Ada F.;Trembleau, Laurent;Botting, Catherine H.;Smith, Margaret C. M.;Jaspars, Marcel;Naismith, James H.
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影响因子：
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通讯作者：
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通讯作者：
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通讯作者：
Mitchell, Douglas A.