A Combined Experimental-Theoretical Study of the LigW-Catalyzed Decarboxylation of 5-Carboxyvanillate in the Metabolic Pathway for Lignin Degradation
A Combined Experimental-Theoretical Study of the LigW-Catalyzed Decarboxylation of 5-Carboxyvanillate in the Metabolic Pathway for Lignin Degradation
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DOI：
10.1021/acscatal.7b01166
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发表时间：
2017-08-01
期刊：
影响因子：
12.9
通讯作者：
Himo, Fahmi
中科院分区：
文献类型：
--
作者：
Sheng, Xiang;Zhu, Wen;Himo, Fahmi
虽然LigW是氨基水解酶超家族的成员，但在细菌木质素降解的代谢途径中，LigW催化5-羧基香草酸酯的非氧化性脱羧基生成香草酸酯。我们现在展示了膜进样质谱仪(MIMS)可用于实时测量瞬时CO2浓度，从而使我们能够确定在LigW催化的反应中，C-C键的断裂产生CO2而不是HCO3-作为初始产物。因此，在pH 7.0下与底物5-羧基香草酸盐孵育LigW会导致最初的二氧化碳生成爆发，随着二氧化碳非酶水合成HCO3-，二氧化碳的生成逐渐减少到平衡值。在酶中同时加入底物和过量的碳酸氢酶，可以完全消除二氧化碳的爆发，说明二氧化碳是初始反应产物。这一发现与密度泛函理论计算的结果完全一致，密度泛函理论计算也支持了C5碳的质子化先于C-C键断裂的机制。限速步骤(C5质子化中间体的形成)的计算势垒为16.8千卡/摩尔，与少神经鞘氨醇单胞菌LigW的观察到的kcat值27相当，后者对应的能垒为16千卡/摩尔。基于MIMS的策略优于确定初始反应产物是CO2还是HCO3-的替代方法，例如使用依赖于pH的染料来监测溶液pH的非常微小的变化。此外，基于MIMS的检测通常适用于所有产生和/或消耗小分子中性气体的酶的研究。
Although it is a member of the amidohydrolase superfamily, LigW catalyzes the nonoxidative decarboxylation of 5-carboxyvanillate to form vanillate in the metabolic pathway for bacterial lignin degradation. We now show that membrane inlet mass spectrometry (MIMS) can be used to measure transient CO2 concentrations in real time, thereby permitting us to establish that C-C bond cleavage proceeds to give CO2 rather than HCO3- as the initial product in the LigW-catalyzed reaction. Thus, incubation of LigW at pH 7.0 with the substrate 5-carboxyvanillate results in an initial burst of CO2 formation that gradually decreases to an equilibrium value as CO2 is nonenzymatically hydrated to HCO3-. The burst of CO2 is completely eliminated with the simultaneous addition of substrate and excess carbonic anhydrase to the enzyme, demonstrating that CO2 is the initial reaction product. This finding is fully consistent with the results of density functional theory calculations, which also provide support for a mechanism in which protonation of the C5 carbon takes place prior to C-C bond cleavage. The calculated barrier of 16.8 kcal/mol for the rate-limiting step, the formation of the C5-protonated intermediate, compares well with the observed kcat value of 27 for Sphingomonas paucimobilis LigW, which corresponds to an energy barrier of 16 kcal/mol. The MIMS-based strategy is superior to alternate methods of establishing whether CO2 or HCO3- is the initial reaction product, such as the use of pH-dependent dyes to monitor very small changes in solution pH. Moreover, the MIMS-based assay is generally applicable to studies of all enzymes that produce and/or consume small-molecule, neutral gases.