Studies on Group 5 Diiron Monooxygenases: Insight into the Mechanism of Regulation of the Diiron Center

Reports: G4 48184-G4: Studies on Group 5 Diiron Monooxygenases: Insight into the Mechanism of Regulation of the Diiron Center

Pablo Sobrado, Virginia Polytechnic Institute and State University

During the reporting period we have been able to advance our understanding of the function of group V bacterial multicomponent monooxygenases. Expression and purification of two of the three proteins in this system have been accomplished. ThmC, the regulatory protein, have been crystallized. However, the crystals that we have obtained diffract very poorly. We have initiated the expression of 15N labeled protein to solve the structure using NMR. ThmD, the oxidoreductase component, has been cloned expressed and thoroughly characterized. This enzyme is unique in that is the only known member of the bacterial multicomponent monooxygenases containing a covalently bound flavin cofactor. A paper describing the unique mechanistic and structural feature of this enzyme was published in the international journal Archives of Biochemistry and Biophysics. We have been using computational modeling and mutagenesis analyses to identify the site of flavin attachment. We have participated in four conferences where results from this research have been presented. One graduate student and four undergraduate students have been working on this project. One manuscript describing the structure of ThmC and one describing the site of flavin attachment will be published.