# Structural Basis for Targeting the Bifunctional Enzyme ArnA

## TL;DR

This study reveals the structural and evolutionary basis of the enzyme ArnA, which helps bacteria resist antibiotics, and proposes new strategies to inhibit its function.

## Contribution

The paper provides a structural and evolutionary analysis of ArnA and proposes novel peptide inhibitors targeting its function.

## Key findings

- ArnA's dehydrogenase and formyltransferase domains evolved independently and fused in Gammaproteobacteria.
- A 2.89 Å cryo-EM structure of ArnA reveals a DH-driven hexameric architecture crucial for activity.
- Structure-based peptide inhibitors were designed to target ArnA's hexamerization and interaction interfaces.

## Abstract

Polymyxin antibiotics are often the last line of defense against multidrug-resistant Gram-negative pathogens. A key resistance mechanism involves the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, mediated by the bifunctional enzyme ArnA. However, the evolutionary rationale and structural basis for ArnA’s domain fusion, hexameric assembly, and catalytic coordination remain mechanistically unresolved. Here, we integrate evolutionary genomics, high-resolution cryo-electron microscopy (cryo-EM), and computational protein design to provide a comprehensive mechanistic analysis of ArnA. Our evolutionary analysis reveals that the dehydrogenase (DH) and formyltransferase (TF) domains evolved independently and were selectively fused in Gammaproteobacteria, suggesting an adaptive advantage. A 2.89 Å cryo-EM structure of apo-ArnA resolves the flexible interdomain linker and reveals a DH-driven hexameric architecture essential for enzymatic activity. 3D variability analysis captures intrinsic conformational dynamics, indicating a molecular switch that may coordinate sequential catalysis and substrate channeling. Structure-based peptide inhibitors targeting the hexamerization and predicted ArnA–ArnB interaction interfaces were computationally designed, offering a novel strategy for disrupting L-Ara4N biosynthesis. These findings illuminate a previously uncharacterized structural mechanism of antimicrobial resistance and lay the groundwork for therapeutic intervention.