# A Dynamic Gate Enables Regioselective Hydroxylation of Free Arginine by a Non‐Canonical Heme Enzyme

## TL;DR

Scientists discovered how a special enzyme can precisely control where it adds oxygen to arginine molecules, which could help make new antibiotics and medicines.

## Contribution

The study reveals a dynamic gating mechanism in a non-canonical heme enzyme that enables programmable regioselective hydroxylation of arginine.

## Key findings

- AglA uses a dynamic loop and key residues to enforce selective hydroxylation at the Nε position of AMPn-Arg.
- Single-site mutations in AglA allow controllable regioselectivity for hydroxylation of free L-arginine.
- Fusing AglA with a reductase improves catalytic efficiency and creates a self-sufficient biocatalytic system.

## Abstract

The YqcI/YcgG family of heme‐dependent enzymes catalyzes guanidine N–H hydroxylation, a critical yet enigmatic step in bioactive natural product biosynthesis. Here, this mechanistic puzzle is resolved through high‐resolution structural snapshots of AglA, a prototypical YqcI/YcgG member, revealing a non‐canonical heme‐binding “sandwich” fold. A dynamic regiochemical gating mechanism is uncovered: substrate‐induced remodeling of loop L2 and key residues (Phe152, Arg179, Phe182) spatially constrains the guanidine group of aminomethylphosphonate‐linked arginine (AMPn‐Arg), enforcing exclusive internal Nε hydroxylation. Single‐site mutations rewire hydrogen‐bond networks to enable hydroxylation of free L‐arginine with controllable regioselectivity (internal Nδ vs terminal Nω) while preserving native internal Nε selectivity for AMPn‐Arg. Crystal structures of engineered variants with free arginine, together with MD simulations, explain how subtle rearrangements of loop L2 and residues Phe152/Arg179/Phe182 pivot the guanidinium group relative to the heme Fe(IV) = O intermediate. Fusing AglA to its native PDR/VanB reductase yields a self‐sufficient chimera with improved catalytic efficiency. This work establishes a structural blueprint for tuning guanidino N–H hydroxylation and demonstrates proof‐of‐principle control of regioselectivity in a non‐canonical heme enzyme, thereby advancing the synthesis of arginine‐based antibiotics and precision‐functionalized therapeutics.

The YqcI/YcgG enzyme AglA employs a unique “sandwich” fold and a dynamic gating mechanism to selectively hydroxylate AMPn‐Arg at Nε. Site‐directed mutations reprogram regioselectivity or enable activity on free arginine by altering hydrogen‐bond networks, precisely positioning substrates. This work establishes a programmable biocatalytic platform for synthesizing arginine‐based antibiotics and functionalized therapeutics.