A New Flavin Redox State in Enzymology

Regents Of The University Of Michigan - Ann Arbor, Ann Arbor MI

Investigators

Abstract

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.