Snapshots of the Catalytic Cycle of an O2, Pyridoxal Phosphate-Dependent Hydroxylase

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts L-arginine to (S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H2O2. To understand this unusual chemistry, we obtain similar to 1.5 angstrom resolution structures that capture intermediates along the catalytic cycle. Our data suggest that RohP carries out a four-electron oxidation and a stereospecific alkene hydration to give the (S)-configured product. Together with our earlier studies on an O-2, PLP-dependent L-arginine oxidase, our work suggests that there is a shared pathway leading to both oxidized and hydroxylated products from L-arginine.