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.