A Dynamic Substrate is Required for MhuD-Catalyzed Degradation of Heme to Mycobilin.
A Dynamic Substrate is Required for MhuD-Catalyzed Degradation of Heme to Mycobilin.
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DOI：
10.1021/acs.biochem.0c00892
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发表时间：
2021-03-30
期刊：
影响因子：
2.9
通讯作者：
Liptak MD
中科院分区：
文献类型：
--
作者：
Thakuri B;O'Rourke BD;Graves AB;Liptak MD
来自结核分枝杆菌的非经典血红素加氧酶MhuD结合一种血红素底物，该底物在平面和非平面褶皱构象之间呈现动态平衡。MhuD通过连续的单加氧和双加氧反应将该底物降解为一种不寻常的分枝杆菌胆素产物。本文建立了血红素底物动力学与MhuD催化的血红素降解之间的因果关系，从而得出一种精细的酶促机制。紫外 - 可见吸收（Abs）和电喷雾电离质谱（ESI - MS）数据表明，有利于褶皱血红素构象形成的第二配位层取代改变了反应的限速步骤，导致单加氧的中 - 羟基血红素中间体可测量的积累。此外，有利于平面底物构象的第二配位层变体的紫外 - 可见吸收和电喷雾电离质谱数据显示，这种变化改变了酶促机制，产生了α - 胆绿素产物。对三种MhuD变体的单周转动力学分析表明，血红素单加氧的速率取决于褶皱底物构象的比例。这些动力学分析还表明，MhuD对中 - 羟基血红素进行双加氧的速率取决于平面底物构象的比例。因此，褶皱血红素构象支持MhuD对血红素进行快速单加氧，但抑制了进一步氧化生成分枝杆菌胆素产物。相反，平面底物构象表现出改变的血红素单加氧区域特异性，随后是中 - 羟基血红素的快速氧化。总之，这些数据得出了MhuD的一种精细的酶促机制，即需要两种底物构象都能参与，才能使三个氧原子快速掺入血红素生成分枝杆菌胆素。
The noncanonical heme oxygenase MhuD from Mycobacterium tuberculosis binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation, resulting in a refined enzymatic mechanism. UV/vis absorption (Abs) and electro-spray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring the population of the ruffled heme conformation changed the rate-limiting step of the reaction, resulting in a measurable buildup of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an α-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled heme conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.
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