Characterization of an isoeugenol monooxygenase (Iem) from Pseudomonas nitroreducens Jin1 that transforms isoeugenol to vanillin

The isoeugenol monooxygenase (iem) gene from Pseudomonas nitroreducens Jin1, responsible for the conversion of isoeugenol to vanillin, was cloned and overexpressed in Escherichia coli. The purified Iem had a predicted molecular mass of 54 kDa. The V_max, KM, and k_cat values for it, using isoeugenol as substrate, were 4.2μmol vanillin min^-1 mg ^-1 of protein, 120μM, and 3.8 s^-1, respectively. Maximum substrate turnover for Iem occurred at 30 °C and pH 9.0. An ¹⁸Oxygen-labeling experiment revealed that oxidative cleavage of isoeugenol by Iem was catalyzed via a monooxygenation reaction, and that incorporation of the oxygen atom from O₂ into vanillin was preferable to incorporation from water. While the catalytic activity of Iem, as prepared, did not require the addition of any organic or metal cofactor, ICP-MS analysis showed 0.7 mol of iron per mol of Iem. Moreover site-directed mutagenesis of Iem of four conserved histidine residues individually, His¹⁶⁷, His ²¹⁸, His²⁸², and His⁴⁷¹, which appear to be involved in ligand bonding with Fe²⁺, resulted in a loss of activity. Enzyme activity was not appreciably influenced by preincubation of Iem with high concentrations of chelators, suggesting that iron is tightly bound. Iem has an iron-mediated mechanism that is widely spread among the three domains of life.