A single monomeric iron center in clavaminate synthase catalyzes three nonsuccessive oxidative transformations

The trifunctional oxygenase clavaminate synthase 2 (CS2) catalyses a hydroxylation reaction and two coupled oxidative reactions, a cyclization and a desaturation, in a nonsuccessive manner. A series of experiments was performed to elucidate the number of CS2 catalytic site(s) utilized in the three oxidative transformations. The stoichiometry of FeII required by CS2 was determined to be one ion per catalytically active enzyme molecule for the cyclization/desaturation reactions, and an affinity label, modeled after the substrate for the hydroxylation reaction, was synthesized and effectively inactivated CS2. The kinetics of this process showed concentration dependence and substrate protection consistent with active site direction. In addition, when this affinity label was incubated with CS2, the enzyme showed the same first-order rate of activity loss over time in both the hydroxylation activity assay and the cyclization/desaturation activity assay. These results support the view that all of the reactions catalysed by CS2 occur in a single catalytic site containing one FeII.