A New Flavin Redox State in Enzymology

酶学中新的黄素氧化还原态

• 批准号: 1508485
• 负责人: Bruce Palfey
• 金额: $ 54万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1508485&HistoricalAwards=false
• 关键词: New; Flavin; Redox; State; Enzymology

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Professor Bruce Palfey from the University of Michigan to explore a newly uncovered flavin redox state. The unexpected and reactive form of vitamin B2 (riboflavin) was recently discovered. This new chemical form has an extra oxygen atom. The extra oxygen atom is necessary for the generation of a compound found in a bacterium that makes numerous antibiotics. Although the chemistry of vitamin B2 has been studied for at least eight decades, how the enzyme could use this oxygen atom, or transfer it to another compound is not known. Also there may be more examples in nature where this newly discovered form of riboflavin is involved. It is also possible that the chemistry catalyzed by this enzyme could be harnessed to accomplish reactions of technological value. The research will address these issues by using a multidisciplinary approach. The investigators engaged in the work will become expert in the latest methods in chemistry and biology, and will help develop web-based curriculum modules that will be useful for undergraduates and graduate students for learning key concepts in biochemistry.The flavin-N5-oxide, a stable over-oxidized form of flavin, is the oxygenating species used by an enzyme in enterocin biosynthesis, EncM. This work will take the first steps in answering questions about the chemistry of the N5 oxide in enzymology. The mechanism of the formation of the N5 oxide on EncM will be studied in detail to test different mechanistic hypotheses, including the possibility of a novel flavin N5-hydroperoxide intermediate. The mechanism of transfer of the oxygen of the N5 oxide of EncM to substrates will also be investigated. Possible mechanisms invoking 1,3 dipolar cycloadditions or radicals will be tested. An understanding of how EncM directs the reaction of molecular oxygen and reduced flavin to the N5 oxide will be obtained through a program of complimentary site-directed mutations to EncM in order to convert it to an oxidase. Conversely, 6 hydroxy-D nicotine oxidase, the closest structural homolog of EncM, will be mutated to convert it into a form that could use the N5 oxide. These investigations will define the biochemistry of the flavin-N5 oxide, and allow for the detection of other enzymes that might use it as an intermediate. This will lay the foundation for mapping the evolution of this unexpected chemistry.

凭借该奖项，化学部的生命过程化学项目正在资助密歇根大学的 Bruce Palfey 教授探索新发现的黄素氧化还原态。 最近发现了维生素 B2（核黄素）的意想不到的活性形式。 这种新的化学形式有一个额外的氧原子。 额外的氧原子对于产生细菌中的一种化合物是必要的，这种化合物可以产生多种抗生素。 尽管人们对维生素 B2 的化学性质进行了至少八年的研究，但这种酶如何利用这个氧原子或将其转移到另一种化合物尚不清楚。 此外，自然界中可能还有更多涉及这种新发现的核黄素形式的例子。 也有可能利用这种酶催化的化学反应来完成具有技术价值的反应。 该研究将通过使用多学科方法来解决这些问题。 从事这项工作的研究人员将成为化学和生物学最新方法的专家，并将帮助开发基于网络的课程模块，这些模块将有助于本科生和研究生学习生物化学的关键概念。黄素-N5-氧化物，黄素的一种稳定的过度氧化形式，是肠菌素生物合成酶 EncM 所使用的氧化物质。 这项工作将迈出回答有关酶学中 N5 氧化物化学问题的第一步。 将详细研究 EncM 上 N5 氧化物形成的机制，以测试不同的机制假设，包括新型黄素 N5-氢过氧化物中间体的可能性。 还将研究 EncM 的 N5 氧化物的氧转移到基材的机制。 将测试调用 1,3 偶极环加成或自由基的可能机制。 通过对 EncM 进行互补定点突变以将其转化为氧化酶，可以了解 EncM 如何引导分子氧和还原黄素反应生成 N5 氧化物。 相反，与 EncM 最接近的结构同源物 6 羟基-D 尼古丁氧化酶将发生突变，将其转化为可以使用 N5 氧化物的形式。这些研究将定义黄素-N5 氧化物的生物化学，并允许检测可能使用它作为中间体的其他酶。这将为绘制这种意想不到的化学反应的演变奠定基础。