Structural analysis of ether bridge forming dioxygenases in nargenicin macrolide biosynthesis Sacha Pidot 1 , Shadi Maghool 1 , Liam Sharkey 2 , Megan Maher 1 1 The University of Melbourne, Australia, 2 Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Australia The nargenicin family of antibiotics are characterised by a narrow spectrum of activity and the presence of an unusual oxa-bridged decalin system. Previous studies have shown that 8,13 ether bridge formation in the best studied member of this family, nargenicin A1, is catalysed by an iron and alpha ketoglutarate dependent dioxygenase (Fe-aKG DD) known as NarN. Fe-aKG DDs use iron and alpha-ketoglutarate as essential co-factors for substrate conversion, producing succinate as part of the reaction. Although the Fe-aKG DD family contains enzymes with a broad range of activities, NarN and the related dioxygenase from streptoseomycin biosynthesis StmO3 (which forms a 9,13 ether bridge), are distinct from all others and sit on a separate branch of the phylogenetic tree. To investigate the structural basis for their unusual biosynthetic activities, we have generated the X-ray crystal structures of both NarN and StmO3. Both enzymes possess remarkable similarity to each other and other well studied members of the Fe-aKG DD family, such as SnoK and SnoN, despite their functional and substrate binding differences. Structural analysis provided key residues that differ between these enzymes and that were predicted to be involved in substrate binding and catalysis. Mutagenesis studies and analysis via luciferase-based succinate formation assay and direct substrate conversion assay showed the impact of each residue on both alpha-ketoglutarate conversion to succinate and product formation. These results expand our understanding of the functionality of Fe-aKG DDs and provide further insight into how these enzymes perform their unusual reactions.
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