Methylamine dehydrogenase (MADH) is an ancient bacterial metabolic enzyme that evolved in a pre-aerobic world. Its anaerobic origins are attested to by the highly evolved multistep chemistry it catalyzes, and its use of ancillary redox protein partners, rather than molecular oxygen, to remove electrons during catalysis. Although a product of divergent evolution, the reductive chemistry catalyzed by the enzyme is analogous to that of the copper-containing amine oxidase family, whose human counterparts are linked to late-diabetic complications and vascular changes in congestive heart disease. MADH contains a novel organic cofactor, tryptophan tryptophylquinone (TTQ), and study of its biogenesis will give new insight into how cofactors have evolved to extend the palette of chemistries enzymes can control: information that could aid in the development of new industrial catalysts, for example. Using a novel combination of single crystal visible spectroscopy, X-ray crystallography and freeze-trapping, this project will probe how the protein controls its complex multistep reaction at atomic resolution by trapping catalytic intermediates in Paracoccus denitrificans MADH (PD-MADH) containing crystals, and determining their structures. Collaborative mutagenesis/structural studies will be used to probe the roles of active site residues in catalysis, substrate binding and TTQ biogenesis. By studying this highly developed metabolic system, a part of the ancient anaerobic redox chemistries still found in mitochondria and chloroplasts today; we can shed light on the fundamental processes of harnessing energy and materials that evolved complex life on earth.
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