Metalloenzymes and metal homeostasis

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Project Summary This research program centers on the bioinorganic chemistry of methanotrophic bacteria, microbes that convert methane, a potent greenhouse gas, to methanol in the first step of their metabolic pathway. As the primary methane sink in nature, methanotrophs are promising tools to mitigate the deleterious effects of global warming on human health, and may be deployed to generate fuels and chemicals from methane in an environmentally-friendly fashion. Moreover, some methanotrophs produce copper-binding natural products that are under investigation as therapeutics. The proposed projects take an integrated biochemical, biophysical, structural, and genetic approach to understanding these processes on the molecular level. The first project addresses the structure and function of particulate methane monooxygenase (pMMO), an integral membrane, copper-dependent enzyme that catalyzes the oxidation of methane to methanol. Despite the availability of pMMO crystal structures and a range of spectroscopic data, the location and atomic details of the copper active site remain unclear, and the sites of substrate, product, and reductant binding have not been elucidated, all prerequisites for elucidating the chemical mechanism. The experimental approach involves characterization of new pMMOs with highly divergent sequences, structural determination of pMMOs in a lipid environment that maintains enzymatic activity, and genetic manipulation of native methanotrophs. The results will lead to a comprehensive understanding of this critically important metalloenzyme and will further understanding of homologs such as ammonia monooxygenase (AMO), another contributor to climate change. The second project focuses on methanobactins (Mbns), ribosomally produced, post-translationally modified natural products secreted by methanotrophs to scavenge copper from the environment. The machinery to biosynthesize and transport Mbns is encoded in Mbn operons, which are also present in a wide range of non- methanotrophic bacteria, suggesting additional functions and unexplored diversity in structure. Mechanistic and structural studies of the core biosynthetic enzyme complex, the iron-containing MbnBC heterodimer, along with characterization of other biosynthetic proteins will be conducted. In addition, the involvement of other operon proteins in release of copper from Mbn will be investigated using both biochemical and in vivo strategies. Taken together, the results will provide new insights into natural products biosynthesis and will impact the use of these molecules as therapeutics for Wilson disease and other disorders of copper metabolism.