Oxygen-activating diiron enzymes have attracted attention for decades for their role in biologically essential and energetically difficult reactions such as the conversion of methane to methanol by methane monooxygenase (MMO) and the desaturation of fatty acids by delta-9-desaturases. Recent work has shown that diiron enzymes also play multiple roles in the biosynthesis of natural product antibiotics. One such enzyme, the aryl-amine oxygenase CmlI, performs the final step in the biosynthesis of the broad-spectrum antibiotic chloramphenicol, converting an arylamine to an arylnitro moiety. Although this type of chemical transformation occurs during the biosynthesis of many antibiotic and cytostatic natural products, CmlI is one of only three well-characterized enzymes known to carry out the six-electron aryl-amine to aryl-nitro oxygenation reaction. The mechanism of aryl-amine oxygenation is poorly understood and several distinct proposals have been advanced. CmlI is an ideal enzyme with which to study this complex mechanism because the exceptional stability of the enzymatic intermediate that preforms substrate oxidation (t<sub>1/2</sub> = 3 hr at 4°C) allows the mechanism of aryl-amine oxidation to be isolated and studied in detail. We have investigated the stoichiometry of the CmlI reaction, the substrate promiscuity of the enzyme, and the identity of the active oxidant in the enzyme mechanism. These studies have been combined with kinetic and spectroscopic experiments to define a novel mechanism for aryl-amine oxidation based on the reactivity of the peroxo intermediate of the reaction cycle. Both chemical and biological implications of this new mechanism will be discussed.

Aryl-amine oxygenation mechanism for a diiron enzyme involved in antibiotic biosynthesis

251st National Meeting (2016)

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