Biosynthesis of Amino Acid Derived Quinone Cofactors

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DESCRIPTION (provided by applicant):Methylamine dehydrogenase (MADH), a metabolic enzyme found in methylotrophic/autotrophic bacteria, contains a quinone cofactor, tryptophan tryptophylquinone (TTQ), derived from the post-translational modification of two Trp residues in the protein. The maturation of MADH involves at least 4 other proteins, and we have begun to characterize one of these proteins, MauG. It is a highly unusual di-heme enzyme responsible for the completion of TTQ synthesis. The natural substrate for MauG (preMADH) is a 119-kDa protein precursor of MADH with a partially formed cofactor. MauG catalyzes a six-electron oxidation to complete TTQ biosynthesis, using either molecular oxygen or hydrogen peroxide as the second substrate. The catalytic reaction involves an unprecedented high-valent di-heme intermediate, which is unusually stable. This application is in response to "NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications." Specific Aim 2 of the parent grant (GM066569) was to solve the crystal structure of MauG in complex with preMADH. This has been achieved to a resolution of 2.1¿, and these crystals can support catalytic turnover to form TTQ without loss of diffraction. The MauG/preMADH structure has revealed features that were not anticipated, and the aims of this competitive revision are built on this new knowledge platform. Using site-directed mutagenesis, X-ray crystallography, kinetic characterization, spectroscopy and mass spectrometry this competitive revision would enable us to rapidly make fundamental discoveries about oxygen activation by a previously unknown high-valent iron intermediate, long range inter-protein electron and radical transfer and mechanisms of oxidative modification to specific amino acid residues within a protein. PUBLIC HEALTH RELEVANCE: MauG is sequentially related to peroxidases that detoxify H2O2 under hypoxic conditions (oxidative stress), but unusually can also activate molecular oxygen, and uses an equivalent catalytic intermediate to human cytochrome P450 enzymes, the major players in drug metabolism, carcinogen activation, biosynthesis of physiologically important molecules, such as steroids, fat-soluble vitamins and fatty acids, as well as the degradation of insecticides and herbicides.