Structural and Functional Characteristics of Potent Dioxygenase from .

Enzymatic C=C double bond cleavage to give carbonyl-species is an emerging alternative to ozonolysis, or stoichiometric use of metal-oxidants. The substrate scope of 4-His Fe dioxygenases, however, appears to be restricted to aromatic compounds with a hydroxy group at the 4-position of the aromatic ring. In-depth structural and functional characterization is a prerequisite to understand and ultimately to extend the substrate scope of this family of enzymes. Herein, DSM 70725 aromatic dioxygenase (ADO) is characterized through X-ray crystallography, biophysical as well as biochemical assays, substrate docking and mutagenesis. ADO features a seven-bladed β-propeller fold and a Fe center coordinated by four histidine residues and shares a conserved structural motif with homologous enzymes despite low sequence identity (<38%). Fe is tightly bound and present in the catalytically active oxidation state at ambient conditions. ADO is robust and retains activity for several freeze/thaw cycles. ADO's interaction with ligands 4-hydroxybenzaldehyde, -vanillin and vanillin indicate that hydrogen-bonding of the phenolic OH group is key to activity. Structural analysis and site-directed mutagenesis indicate that two key residues (Y136 and K169), and the substrate's hydroxy group, are essential for accurately positioning the double bond toward the activated oxygen at the Fe center. ADO wild-type exhibits the highest reported activity for converting isoeugenol to vanillin (231 μmol min mg).