AmericanChemicalSociety.com

During the reporting period we have been able to significantly advance or research on group V bacterial multicomponent monooxygenasses.  Expression and purification of two of the three proteins in this system have been accomplished. ThmC, the regulatory protein, have been crystallized and the crystal diffracts at 3.0 A resolution. We are currently in the process of solving the 3D-structure.  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. ThmD was expressed in E. coli as a fusion to maltose binding protein (MBP) and isolated to homogeneity after removal of the MBP. Purified ThmD contains covalently bound FAD, [2Fe-2S] center, and was shown to use ferricyanide, cytochrome c, 2,6-dichloroindophenol, and to a lesser extent, oxygen as surrogate electron acceptors. ThmD displays 160-fold preference for NADH over NADPH and functions as a monomer. The flavin-binding domain of ThmD (ThmD-FD) was purified and characterized.  ThmD-FD displayed similar activity as the full-length ThmD and showed a unique flavin spectrum with a major peak at 463 nm and a small peak at 396 nm. Computational modeling and mutagenesis analyses suggest a novel three-dimensional fold or covalent flavin attachment in ThmD. We have participated in three conferences where results from this research have been presented. One graduate student and two undergraduate students have been working on this project. One manuscript is currently under review.