# Structural basis for iterative methylation by a cobalamin-dependent radical S-adenosylmethionine enzyme in cystobactamids biosynthesis

## TL;DR

This study reveals how a specific enzyme performs multiple methylations using two different mechanisms, providing structural insights into its function.

## Contribution

The study identifies a key residue that controls SAM binding conformations for cobalamin methylation and iterative substrate methylation.

## Key findings

- Trp271 regulates a mobile loop to allow SAM to bind in two conformations for different methylation steps.
- Structures of CysS show how the enzyme accommodates substrates and products during iterative methylation.
- The enzyme uses a ping–pong mechanism with two SAM molecules for each methylation step.

## Abstract

Cbl-dependent radical SAM methylases (RSMs) use two SAM molecules to methylate unactivated carbons. One SAM is used to methylate cob(I)alamin, generating methylcobalamin (MeCbl). A second SAM molecule is used to generate a 5′-deoxyadenosyl radical (5′dA•). Although three structures of Cbl-dependent RSMs have been determined, none provide insight into how SAM methylates cob(I)alamin. In this work, we demonstrate that Trp271 regulates the positioning of a mobile loop, enabling SAM to bind in two distinct conformations, one of which is suitable for cob(I)alamin methylation. Our structures of CysS, two with substrates/products bound and one in the absence of a substrate or product, also provide insight into how a single cobalamin-dependent RSM can perform multiple methylations on one substrate.

Cystobactamids are nonribosomal peptide natural products that function as DNA gyrase inhibitors, exhibiting significant antibacterial activity. They are isolated from Cystobacter sp. Cbv34 and contain various alkoxy groups on para-aminobenzoic acid moieties, which are believed to play a crucial role in antibacterial functions. The alkoxy groups are generated by iterative methylations on a methoxy group by the cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzyme CysS. CysS catalyzes up to three methylations to give ethoxy, isopropoxy, sec-butoxy, and tert-butoxy groups. For each methylation, CysS uses a ping–pong mechanism in which two molecules of SAM are consumed. One SAM is used to methylate cob(I)alamin, while another generates a 5′-deoxyadenosyl 5′-radical to initiate substrate methylation. However, little is known about how the enzyme promotes both Cbl methylation and iterative substrate methylation, which occur by polar SN2 and radical processes, respectively. Here, we report three X-ray crystal structures of a homolog of CysS from Corallococcus sp. CA054B. Two were determined in the presence of methoxy- and ethoxy-containing substrates, showing how CysS accommodates substrates and products during iterative methylation. The third structure, determined in the absence of a substrate, exhibits structural changes that reorient the SAM’s conformation to allow for the methylation of cob(I)alamin.