Journal Article

A highly dynamic mononuclear non-heme iron enzyme for the two-step isonitrile biosynthesis

Zhang, Wenjun, Drennan, Catherine L., Ye, Naike, Del Rio Flores, Antonio

2026

Overview

The recent discovery of the isonitrile biosynthetic enzyme ScoE expanded the catalytic repertoire of the Fe(II)/αKG-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 αKG 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 αKG-bound structure, and a structure with both CADA and αKG 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 αKG cleavage products for a second molecule of αKG. 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. Sixteen crystal structures of the non-heme iron enzyme Rv0097 reveal how protein motions are orchestrated in the two-step, α-ketoglutarate-dependent, biosynthesis of isonitrile functional groups from substrate glycyl moieties.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/45/173 / 631/45/535/1266 / 639/638/45/607/1168 / 82/80 / Acids / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Binding sites / Biosynthesis / Cleavage / Crystal structure / Crystallography, X-Ray / Dioxygenase / Enzymes / Functional groups / Heme / Humanities and Social Sciences / Iron / Iron - chemistry / Iron - metabolism / Ketoglutaric acid / Ketoglutaric Acids - metabolism / Models, Molecular / multidisciplinary / Mycobacterium tuberculosis - enzymology / Mycobacterium tuberculosis - genetics / Natural products / Nitriles - chemistry / Nitriles - metabolism / Oxidation / Reaction intermediates / Science / Science (multidisciplinary) / Site-directed mutagenesis / Tuberculosis