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.

有了这个奖项，化学部门的化学过程课程正在为密歇根大学的Bruce Palfey教授提供资金，以探索新发现的Flavin氧化还原州。 最近发现了维生素B2（核黄素）的意外和反应性形式。 这种新的化学形式具有额外的氧原子。 额外的氧原子对于生成多种抗生素的细菌中发现的化合物是必需的。 尽管已经研究了至少八十年的维生素B2的化学，但酶如何使用该氧原子，或将其转移到另一种化合物中。 同样，这种新发现的核黄素形式可能还有更多例子。 还可以利用该酶催化的化学作用来实现技术价值的反应。 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，并将有助于开发基于Web的课程模式，这将对本科生和研究生学习生物化学中的关键概念。 这项工作将在回答有关酶学中N5氧化物化学的问题时采取第一步。 将详细研究ENCM上N5氧化物形成的机制，以检验不同的机械假设，包括新的黄素N5-氢过氧化物中间体的可能性。 还将研究ENCM的N5氧化物氧向底物转移的机理。 将测试调用1,3偶极环加成或自由基的可能机制。 了解ENGM如何通过一个免费位置定向突变的程序对ENCM的程序获得了分子氧和减少黄素对N5氧化物的反应，以将其转化为氧化酶。 相反，将将6羟基-D尼古丁氧化酶（最接近ENCM的结构同源物）变异，以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，并允许检测可能将其用作中间体的其他酶。这将为绘制这种意外化学的演变奠定基础。