Structural Characterization of the l-Homo-Tyrosine β-Hydroxylase EcdG Involved in Echinocandin Biosynthesis Guides the Engineering of Its Homologous Protein GloM

β-Hydroxy-amino acids are key structural motifs of many bioactive natural products, but enzymatic β-hydroxylation of an aromatic amino acid appears to be uncommon. In known cases, this reaction typically occurs on a residue residing in a peptide scaffold or tethered on a carrier protein via a thioester linkage. EcdG, an α-ketoglutarate/Fe(II)-dependent dioxygenase involved in the biosynthesis of echinocandins, is an exception, catalyzing the stereoselective β-hydroxylation of a free l-homotyrosine amino acid. Here, we report a comprehensive structural and mechanistic study of EcdG and use knowledge gained from this study for functional modulation of the homologous protein GloM. High-resolution crystal structures of EcdG in complex with α-ketoglutarate and l-homotyrosine were determined. With the association of site-directed mutagenesis, the molecular basis for substrate recognition and the regio- and stereoselectivity of EcdG was revealed and then used to guide the engineering of GloM. The substrate specificity and oxidation capability of this homologous protein were altered, enabling selective transformation of the unnatural substrate l-homophenylalanine to either a β-hydroxyl product or a γ-keto product. This work provides mechanistic insights into a type of uncommon β-hydroxylase and expands the enzymatic toolbox for selective oxidative functionalization of a free aromatic amino acid.