A New Flavin Redox State in Enzymology

通过这一奖项，化学系的生命过程化学计划资助密歇根大学的布鲁斯·帕尔菲教授探索新发现的黄素氧化还原状态。最近发现了一种意想不到的活性形式的维生素B2(核黄素)。这种新的化学形式有一个额外的氧原子。额外的氧原子是产生一种化合物所必需的，这种化合物存在于制造多种抗生素的细菌中。虽然对维生素B2的化学研究已经至少有80年了，但这种酶如何利用这种氧原子，或者将其转移到另一种化合物中，还不清楚。此外，自然界中可能有更多的例子涉及这种新发现的核黄素形式。这种酶催化的化学反应也有可能被利用来完成具有技术价值的反应。这项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-氧化物是黄素的一种稳定的过氧化形式，是肠粘素生物合成中的一种酶使用的氧化物种EncM。这项工作将是回答有关N5氧化物的酶化学问题的第一步。将详细研究N5氧化物在EncM上的形成机理，以检验不同的机理假说，包括一种新的黄素N5-过氧化氢中间体的可能性。还将对EncM的N5氧化物的氧转移到衬底的机理进行研究。将测试引发1，3偶极环加成或自由基的可能机制。了解EncM如何引导分子氧和黄素还原为N5氧化物的反应，将通过对EncM进行互补的定点突变程序来获得，以便将其转化为氧化酶。相反，EncM最接近的结构同系物6-羟基-D尼古丁氧化酶将发生突变，将其转化为一种可能使用N5氧化物的形式。这些研究将确定黄素-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。这将为绘制这种意想不到的化学物质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.