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

## TL;DR

This paper reveals how the enzyme Rv0097 performs a two-step chemical reaction to create isonitrile groups using structural snapshots and mutagenesis data.

## Contribution

The study provides new structural and mechanistic insights into the two-step isonitrile biosynthesis by a non-heme iron enzyme.

## Key findings

- Sixteen crystal structures of Rv0097 show conformational changes during the isonitrile biosynthesis process.
- The enzyme uses α-ketoglutarate cleavage in two half-reactions to form isonitriles from glycyl-fatty acid substrates.
- Structural and mutagenesis data explain the enzyme's preference for substrates with long alkyl chains.

## Abstract

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.