The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para‐hydroxybenzoate catabolism
The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para‐hydroxybenzoate catabolism
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DOI：
10.1111/mmi.14094
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发表时间：
2018-10
影响因子：
3.6
通讯作者：
Huan Zhao;Ying Xu;Shuangjun Lin;J. Spain;N. Zhou
中科院分区：
文献类型：
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作者：
Huan Zhao;Ying Xu;Shuangjun Lin;J. Spain;N. Zhou
NIH迁移是一种化学重排现象，在此过程中，芳香环上的取代基会发生分子内迁移，这一现象主要发生在酶促羟基化反应中。40年来，羧基的NIH迁移的分子机制一直是个谜。在此，我们阐明了对羟基苯甲酸（PHB）转化为龙胆酸（GA，即2,5 - 二羟基苯甲酸）过程中该反应的分子机制。来自利用PHB的侧孢短芽孢杆菌PHB - 7a的三个基因（phgABC）分别编码对羟基苯甲酰辅酶A连接酶、对羟基苯甲酰辅酶A羟化酶和龙胆酰辅酶A硫酯酶，这些酶通过一条涉及辅酶A硫酯形成、羟基化同时伴随乙酰辅酶A部分的1,2 - 迁移以及硫酯水解的途径，催化PHB转化为GA。通过对异源表达的phgABC进行稳定同位素实验，明确证实了羧基的迁移，即分别将2,3,5,6 - 四氘代 - PHB和[羧基 - ¹³C] - PHB转化为3,4,6 - 三氘代 - GA和[羧基 - ¹³C] - GA。这与氢和乙酰基取代基的NIH迁移不同，后者由单一加氧酶催化反应，且不涉及硫酯。这种羧基迁移的三步策略的发现揭示了辅酶A硫酯在生物化学中的新作用，同时也说明了微生物在碳循环中分解代谢的多样性和复杂性。
The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para‐hydroxybenzoate (PHB) to gentisate (GA, 2, 5‐dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB‐7a encode enzymes (p‐hydroxybenzoyl‐CoA ligase, p‐hydroxybenzoyl‐CoA hydroxylase and gentisyl‐CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2‐shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6‐tetradeutero‐PHB and [carboxyl‐13C]‐PHB to 3, 4, 6‐trideutero‐GA and [carboxyl‐13C]‐GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three‐step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.