A New Way of Belonging: Active-Site Investigation of L-DOPA Dioxygenase, a VOC Family Enzyme from Lincomycin Biosynthesis
A New Way of Belonging: Active-Site Investigation of L-DOPA Dioxygenase, a VOC Family Enzyme from Lincomycin Biosynthesis
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一种新的归属方式：L-DOPA 双加氧酶（林可霉素生物合成中的 VOC 家族酶）的活性位点研究
DOI：
10.1021/acs.biochem.9b00456
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Hoffmann, Thomas W.
中科院分区：
文献类型：
--
作者：
Colabroy, Keri L.;Horwitz, Alyssa D.;Basciano, Victoria R.;Fu, Yizhi;Travitz, Kelly M.;Robinson, Miranda K.;Shimanski, Brittany A.;Hoffmann, Thomas W.
外源性双加氧酶对邻苯二酚的分解是必不可少的。最大的儿茶酚或1，2-二羟基苯的天然储存库是植物木质组织聚合物木质素。邻位氧螯合双加氧酶(VoC)是一类重要的外加氧双加氧酶，大多数是降解天然和人造芳香环的分解代谢途径的一部分，而L-多巴(L-3，4-二羟基苯丙氨酸)双加氧酶是一种参与天然产物生物合成的VOC酶。所有VOC超家族成员都具有保守的催化元件，然而，尽管对VOC酶进行了数十年的研究，但VOC结构域结构与酶功能之间的关系仍然复杂且知之甚少。在此，我们提出了L-多巴双加氧酶是一类新的VOC外醇双加氧酶的代表性成员的证据。根据其与乙氧基酶的进化相似性，我们通过诱变、动力学和pH研究对链霉菌L-DOPA双加氧酶(LmbB1)的活性部位进行了仔细的研究。我们的结果表明，L-多巴双加氧酶反应依赖于活性部位的酪氨酸和组氨酸，并且即使在铁连接残基上也具有显著的抗突变能力。对作为pH函数的切割反应的评价支持组氨酸在酸碱催化中的作用。活性中心的结构在功能上与现有的VOC外醇双加氧酶催化知识是一致的。
Extradiol dioxygenase chemistry is essential for catechol breakdown. The largest natural reservoir of catechols, or 1,2-dihydroxybenzenes, is the plant woody-tissue polymer lignin. Vicinal–oxygen–chelate (VOC) dioxygenases make up the largest group of characterized extradiol dioxygenases, and while most are found as part of catabolic pathways degrading a variety of natural and human-made aromatic rings, L-DOPA (l-3,4-dihydroxyphenylalanine) dioxygenase is a VOC enzyme that participates in the biosynthesis of a natural product. All VOC superfamily members shared conserved elements of catalysis, yet despite decades of investigation of VOC enzymes, the relationships between VOC domain architecture and enzymatic function remain complex and poorly understood. Herein, we present evidence that L-DOPA dioxygenase is the representative member of a new topological class of VOC extradiol dioxygenases. Guided by its evolutionary similarity to glyoxylase enzymes, we performed a careful investigation of theStreptomyces lincolnensisL-DOPA dioxygenase (LmbB1) active site through mutagenesis, kinetic, and pH studies. Our results demonstrate that the L-DOPA dioxygenase reaction depends upon an active-site tyrosine and histidine and is remarkably resilient to mutation, even at the iron-ligating residues. Evaluation of the cleavage reaction as a function of pH supports the role of a histidine in acid–base catalysis. The active-site architecture is functionally consistent with the existing knowledge of VOC extradiol dioxygenase catalysis.