Roles of protein structure and diiron cluster chemistry in oxygen activation

描述（由申请人提供）：通过含二铁簇的酶激活O2以插入生物分子通常需要结合并激活O2的簇与以多种方式指导和调节该过程的蛋白质之间的伙伴关系。充分研究的二铁单加氧酶都使用富含羧酸根的二铁簇配体结构，每个铁有一个His配体和一个4螺旋束蛋白质折叠。此外，委员会认为， 大多数催化反应如C-H或芳族羟基化，它们是机械相关的反应。为了解决二铁簇和蛋白质结构在O2活化中的作用，并进一步探索二铁簇加氧酶可获得的化学范围，需要新类别的二铁加氧酶，其：（i）在不同的蛋白质环境中呈现二铁核心，（ii）引发新的核心结构，和/或（iii）催化新的反应。在研究链霉菌氯霉素的生物合成途径时，我们发现了两种新的双铁酶，可以满足这些要求。第一种酶，CmlA，是一类至少50种未表征的酶的创始成员，这些酶催化必需的？抗生素、生物抑制剂和化疗剂的羟基化，因为它们在基于非核糖体肽合成酶（NRPS）的途径中合成。序列同源性、光谱学和产物分析表明，CmlA是第一个被识别的二铁单加氧酶：（i）结合新的？内酰胺酶折叠，（ii）催化B-羟基化，和（iii）使每个Fe具有一个以上His配体。我们已经在使用各种光谱的初步研究中表明，CmlA的Fe（II）Fe（II）状态仅当其与在其巯基化结构域上共价负载有氯霉素前体L-p-NH 2-苯丙氨酸（PAPA）的NRPS（CmlP）络合时才与O2反应。第二种新的二铁酶，CmII，催化氯霉素生物合成的最后一步，芳香胺转化为硝基。这种化学性质直到最近才在二铁家族中被认识到，并且特征很差。初步研究表明，CmlI使用4-螺旋束折叠，但与CmlA一样，它每个Fe有一个以上的His配体。与CmlA不同，它不会催化任何常见的二铁单加氧酶反应，并且它将接受其底物游离或与CmlP结合。我们建议在结构上表征CmlA和CmlI与和没有他们的基板。这两种酶的有效的单周转系统将允许瞬态动力学技术被用来搜索和捕获反应循环的光谱表征中间体。替代底物和活性位点诱变将用于探测这两种酶的分子机制。光谱标记将用于揭示与CmlP的相互作用区，以研究该组分调节O2结合的方法。调节的中间体和模式将直接与本实验室长期研究的甲烷单加氧酶的中间体和模式进行比较。这项工作将对O2激活和调节的基本机制产生影响，并可能导致对新型抗生素生产策略的重要见解。

DESCRIPTION (provided by applicant): The activation of O2 by diiron cluster-containing enzymes for insertion into biological molecules usually requires a partnership between the cluster, which binds and activates O2, and the protein, which directs and regulates the process in a variety of ways. The well-studied diiron monooxygenases all use carboxylate-rich diiron cluster ligand structures with one His ligand per iron and a 4-helix-bundle protein fold. Moreover, most catalyze reactions such as C-H or aromatic hydroxylation, which are mechanistically related reactions. In order to resolve the roles of the diiron cluster and protein structure in O2 activation, and to further explore the range of chemistry accessible by the diiron cluster oxygenases, new classes of diiron oxygenases are needed that: (i) present the diiron core in a different protein environment, (ii) invoke new core structures, and/or (iii) catalyze novel reactions. While studying the biosynthetic pathway for chloramphenicol from Streptomyces, we found two new diiron enzymes that address these requirements. The first enzyme, CmlA, is the founding member of a class of at least 50 uncharacterized enzymes that catalyze essential ?-hydroxylation of antibiotics, biostatics and chemotherapy agents, as they are synthesized in nonribosomal peptide synthetase (NRPS)-based pathways. Sequence homology, spectroscopy, and product analysis show that CmlA is the first diiron monooxygenase recognized to: (i) bind the diiron cluster in a novel ?-lactamase fold, (ii) catalyze b- hydroxylation, and (iii) incorporae more than one His ligand per Fe. We have shown in preliminary studies using a variety of spectroscopies that the Fe(II)Fe(II) state of CmlA reacts with O2 only when it is complexed with its NRPS (CmlP) covalently loaded with the chloramphenicol precursor L-p-NH2-phenylalanine (PAPA) on its thiolation domain. The second new diiron enzyme, CmlI, catalyzes the final step in chloramphenicol biosynthesis, aromatic amine to nitro conversion. This chemistry has only recently been recognized in the diiron family and is poorly characterized. Preliminary studies show that CmlI uses the 4-helix bundle fold, but, like CmlA, it has more than one His ligand per Fe. Unlike CmlA, it will not catalyze any of the common diiron monooxygenase reactions, and it will accept its substrate either free or bound to CmlP. We propose to structurally characterize CmlA and CmlI with and without their substrates. Efficient single turnover systems for both enzymes will allow transient kinetic techniques to be used to search for and trap reaction cycle intermediates for spectroscopic characterization. Alternative substrates and active site mutagenesis will be used to probe the molecular mechanisms of both enzymes. Spectroscopic labels will be used to reveal the interaction zones with CmlP to investigate the means by which this component regulates O2 binding. Both the intermediates and modes of regulation will be directly compared with those of methane monooxygenase, long studied in this laboratory. This work will bear on fundamental mechanisms of O2 activation and regulation as well as potentially leading to important insights into strategies for the production of novel antibiotics.