Structure determination of reaction intermediates in macromolecular complexes

本研究的目的是确定两种不同反应中反应中间体的结构， 在大分子复合物中起作用的金属酶系统。第一个具体目标将决定 氧介导的Ib类核糖核苷酸还原酶活化的中间体结构，仅在 原核生物，最近发现采用一种新的二锰（III）-酪氨酰基自由基辅因子的催化。 该项目将在K99资助期间完成，并将在早期进行晶体学表征。 反应中间体通过冷冻捕获和诱变技术。后期中间体将稳定 通过利用反应的pH和温度依赖性及其对同位素效应的敏感性。 晶体中反应中间体的光谱表征将提供独立的验证， 结构.酶的本质及其作为脱氧核苷酸的主要模式的功能 在许多人类病原体中的产生使其活化反应成为一种可能的新途径， 抗生素开发第二个目标将探索RNA甲基化酶的底物结合结构， 一种[4Fe-4S]簇，S-腺苷-L-甲硫氨酸（SAM）辅因子，用于催化一种新的甲基转移机制 在未活化的碳中心的反应。待研究的酶（大肠杆菌RlmN） 甲基化赋予调节肽基转移酶中心内的翻译的能力的位置， 核糖体的大亚基。RlmN与甲基化酶（金黄色葡萄球菌Cfr）相关， 稍有不同的位点选择性。Cfr介导的23 S核糖体甲基化与抗 针对PTC的抗生素。RlmN和Cfr靶向23 S亚基内的特异性腺嘌呤位点，并且是最常见的。 在核糖体的大片段中是有活性的。拟议工作的目标是获得结构性 关于RlmN与其底物的最小和越来越大的片段结合的信息，并研究 捕获的反应中间体的结构。这项工作将在K99资助期间开始， 在独立阶段继续。了解酶与其底物结合的结构， 在反应途径中的各种状态将提供有关结构基础的关键信息， 为阐明Cfr耐药性的进化机制和特异性奠定基础。

The objective of the proposed research is to determine structures of reaction intermediates in two separate metalloenzyme systems that operate within macromolecular complexes. The first specific aim will determine structures of intermediates in oxygen-mediated activation of class Ib ribonucleotide reductase, found only in prokaryotes and recently discovered to employ a novel dimanganese(III)-tyrosyl radical cofactor for catalysis. This project will be completed during the K99 funding period and will crystallographically characterize early reaction intermediates via freeze trapping and mutagenesis techniques. Later intermediates will be stabilized by exploiting the pH and temperature dependence of the reaction and its susceptibility to isotope effects. Spectroscopic characterization of reaction intermediates in the crystal will provide independent verification of structures. The essential nature of the enzyme and its function as the primary mode of deoxynucleotide production in a number of human pathogens makes its activation reaction a possible new avenue for novel antibiotic development. The second aim will explore substrate-bound structures of an RNA methylase that uses a [4Fe-4S] cluster, S-adenosyl-L-methionine (SAM) cofactor to catalyze a mechanistically novel methyl transfer reaction at an unactivated carbon center. The enzyme to be studied (Escherichia coli RlmN) specifically methylates a position that imparts the capacity to modulate translation within the peptidyl transferase center of the large subunit (23S) of the ribosome. RlmN is related to a methylase (Staphylococcus aureus Cfr) with a slightly different site selectivity. Cfr-mediated methylation of the 23S ribosome is implicated in resistance to antibiotics that target the PTC. RlmN and Cfr target a specific adenine site within the 23S subunit and are most active in the context of large fragments of the ribosome. The goal of the proposed work is to gain structural information about RlmN bound to minimal and increasingly large fragments of its substrate and to investigate the structures of trapped reaction intermediates. This work will begin during the K99 funding period and will continue during the independent phase. Understanding the structure of the enzyme bound to its substrate and at various states in the reaction pathway will provide critical information about the structural basis for mechanism and specificity and will lay the foundation to elucidate evolution of antibiotic resistance in Cfr.