Biosynthesis of Amino Acid Derived Quinone Cofactors

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DESCRIPTION (provided by applicant): Found in all kingdoms of life, copper-containing amine oxidases (CuAOs) contain two redox centers. One is a copper ion, and the other is a novel organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), derived from the post-translational modification of a Tyr residue in the protein. The biosynthesis of TPQ is an auto-catalytic process, which is molecular oxygen and cupric ion dependent. Formation of such self- processed cofactors likely played a key role in enzyme evolution, as they extended the chemical functionalities available for catalysis without the evolution of separate biosynthetic enzymes. Methylamine dehydrogenase (MADH), a metabolic enzyme found in methylotrophic/autotrophic bacteria, also contains a quinone cofactor, tryptophan tryptophylquinone (TTQ), derived from the post-translational modification of two Trp residues in the protein. In contrast to CuAOs, the maturation of MADH involves at least 4 other proteins. In the previous grant period, we have begun to characterize one of these proteins, MauG. It is a highly unusual diheme enzyme responsible for the completion of TTQ synthesis, which can use either molecular oxygen or hydrogen peroxide as a substrate. In the last few years, the list of enzymes containing amino acid derived cofactors has grown rapidly. In addition, both TPQ and TTQ synthesis require oxygen substrates, but in very different reactions. The ability to activate molecular oxygen underpins all aerobic biology, but the details of how this is achieved are still poorly understood. I propose to use the yeast Hansenula polymorpha methylamine oxidase (HPAO) to study the structural basis of TPQ formation. Through a novel combination of single crystal UV/visible microspectrophotometry, X-ray crystallography and anaerobic / aerobic freeze trapping, reaction intermediates in biogenesis will be trapped in the crystal. The controversial role of copper in CuAO will be explored through metal replacement studies. In addition, I propose to explore MADH maturation through X-ray crystallographic studies of MauG, in particular focusing on the atypical behavior of the c-type heme(s).PUBLIC HEALTH RELEVANCE: The aberrant actions of human copper-containing amine oxidases are linked to congestive heart disease, late-diabetic complications and Alzheimer's disease. One human copper-containing amine oxidase is involved in the inflammatory response, and represents a new target for anti-inflammatory drugs. MauG is sequentially related to peroxidases that detoxify H2O2 under hypoxic conditions (oxidative stress), but unusually can also activate molecular oxygen with mechanistic similarities 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.