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

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 N ε hydroxylation. Single‐site mutations rewire hydrogen‐bond networks to enable hydroxylation of free L‐arginine with controllable regioselectivity (internal N δ vs terminal N ω ) while preserving native internal N ε 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.