A New Flavin Redox State in Enzymology

凭借这一奖项，化学部门的生命过程化学项目资助密歇根大学的Bruce Palfey教授探索新发现的黄素氧化还原状态。维生素B2（核黄素）的意外反应形式是最近发现的。这种新的化学形式多了一个氧原子。多余的氧原子对于细菌中产生的一种化合物是必要的，这种化合物可以制造许多抗生素。尽管人们对维生素B2的化学性质已经进行了至少80年的研究，但这种酶是如何利用这个氧原子，或将其转移到另一种化合物上的，目前还不清楚。此外，在自然界中可能还有更多的例子涉及到这种新发现的核黄素形式。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。本研究将采用多学科方法解决这些问题。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，这些课程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 -氧化物是黄素的一种稳定的过度氧化形式，是肠球蛋白生物合成过程中一种酶所使用的氧合物质。这项工作将在回答酶学中N5氧化物的化学性质问题方面迈出第一步。我们将详细研究N5氧化物在EncM上形成的机制，以测试不同的机制假设，包括一种新的黄素N5-氢过氧化物中间体的可能性。研究了enm中N5氧化物的氧向衬底转移的机理。将测试调用1,3偶极环加成或自由基的可能机制。通过对EncM进行互补位点定向突变以将其转化为氧化酶，可以了解EncM如何指导分子氧和还原黄素与N5氧化物的反应。相反，与EncM结构最接近的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，并允许检测其他可能将其用作中间体的酶。这将为绘制这种意想不到的化学的进化图谱奠定基础。

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.