Methylation in Antibiotic Biosynthesis: Methylcobalamin-Radical SAM Enzymes

简介（由申请人提供）：我于2000年获得塞尔维亚和黑山贝尔格莱德大学化学学士学位，2005年5月获得伊利诺伊大学厄巴纳-香槟分校有机化学博士学位。在研究生学习期间，我在David Y. Gin和Wilfred A. van der Donk教授的共同指导下开发了化学选择性碳水化合物-肽连接的新方法。目前，我是哈佛医学院Christopher T. Walsh教授实验室的Damon Runyon癌症研究基金会博士后。HMS和沃尔什教授提供了一个优秀的研究环境，并致力于博士后的成功。我的博士后研究重点是最近发现的一类非血红素Fe （II）卤化酶的表征，能够对非活性炭中心进行卤化。到目前为止，我们已经重建了巴巴胺体系的卤化活性。在这项研究中，我们证明了载体蛋白拴链l -亮氨酸底物的未激活甲基的三氯化是由两种非血红素类卤化酶BarB1和BarB2的串联作用介导的。我目前正在研究非活性炭中心卤化的机理方面，通过研究预稳态动力学参数和催化过程中金属中心的EPR和穆斯堡尔研究。拟建项目的目的是对抗生素生物合成中进行sp2碳中心甲基化的甲基钴胺基SAM酶的机制描述。我们的目标是了解自然界用来引导甲基钴胺素、铁硫簇和脱氧腺苷基自由基在酶学中进行这种新型碳-碳键形成的逻辑。甲基化事件将在氨基香豆素抗生素生物合成中的5-甲基吡咯-2-羧酸药效团的产生和β -内酰胺抗生素硫霉素生物合成中的羟乙基侧链的产生中进行研究。更好地了解参与抗生素生物合成的酶可以通过组合生物合成开发新的抗生素变体。这一点尤其重要，因为细菌对常用抗生素产生了耐药性。

DESCRIPTION (provided by applicant): I have obtained B.Sc. in chemistry from University of Belgrade, Serbia and Montenegro, in 2000, and Ph.D. in organic chemistry from University of Illinois at Urbana-Champaign in May 2005. During graduate studies, I have developed new methods for the chemoselective carbohydrate-peptide ligations under the joint guidance of Professors David Y. Gin and Wilfred A. van der Donk. Currently, I am a Damon Runyon Cancer Research Foundation postdoctoral fellow in the laboratory of Professor Christopher T. Walsh at Harvard Medical School. HMS and Professor Walsh is providing an outstanding research environment and is committed to the success of the postdoctoral fellows. My postdoctoral research is focused on the characterization of a recently discovered class of nonheme Fe (II) halogenases, capable of carrying out halogenation of unactivated carbon centers. Thus far, we have reconstituted halogenation activity in the barbamide system. In this study, we demonstrated that the triple chlorination of the unactivated methyl group of the carrier-protein tethered L-leucine substrate is mediated by the tandem action of two nonheme Fell halogenases, BarB1 and BarB2. I am currently investigating mechanistic aspects of halogenation of unactivated carbon centers through the investigation of pre-steady state kinetic parameters and EPR and Mossbauer investigation of metal center during the catalysis. The objective of the proposed project is mechanistic description of methylcobalamin-radical SAM enzymes that carry out methylations of sp2 carbon centers in antibiotic biosynthesis. Our goal is to understand the logic that nature uses to channel methylcobalamin, iron-sulfur clusters and deoxyadenosyl radicals to perform this novel carbon-carbon bond formation in enzymology. The methylation event will be studied in the context of generation of the 5-methylpyrrole-2-carboxylate pharmacophore in aminocoumarin antibiotic biosynthesis, and hydroxyethyl side chain in the biosynthesis of beta lactam antibiotic thienamycin. Better understanding of enzymes involved in the antibiotic biosynthesis can lead to the development of new antibiotic variants through combinatorial biosynthesis. This is especially important because of the development of bacterial resistance to commonly used antibiotics.