Abstact

The recent discovery of the isonitrile biosynthetic enzyme ScoE expanded the catalytic repertoire of the Fe(II)/alphaKG-dependent dioxygenase enzyme family. ScoE synthesizes an isonitrile functional group from a glycyl-fatty acid adduct, with both the isonitrile nitrogen and carbon atoms coming from the glycyl moiety. This challenging chemistry cannot be performed in a single step. Instead, the mechanism appears to require two half reactions, each involving alphaKG cleavage to generate a highly reactive iron-oxygen species. Here, we report sixteen crystal structures that provide snapshots along the reaction trajectory of Rv0097, a ScoE homolog from Mycobacterium tuberculosis. These structures, which are both of wild-type and Rv0097 variants, include a substrate 3-((carboxymethyl)amino)decanoic acid (CADA)-bound structure, an alphaKG-bound structure, and a structure with both CADA and alphaKG bound. These structural data reveal how Rv0097 employs conformational rearrangements to protect the unstable CADA-reaction intermediate that is formed in the first half reaction while swapping out alphaKG cleavage products for a second molecule of alphaKG. Additionally, these structures, together with data from site-directed mutagenesis, provide insight into Rv0097's preference for substrates with long alkyl chains, potentially facilitating efforts to re-engineer ScoE/Rv0097 to synthesize isonitrile functional groups on a wider range of small molecules.