Snapshots of the catalytic cycle of an O2, pyridoxal phosphate‐dependent hydroxylase

Abstract

The organic cofactor pyridoxal phosphate (PLP) is used by enzymes to catalyze numerous modifications of amino acid substrates. Recently, the diversity of PLP‐dependent enzymes has been expanded with the discovery of several enzymes that use O2 as a co‐substrate to perform chemically challenging oxidative reactions. The interesting chemistry catalyzed by such O2‐, PLP‐dependent oxidases prompted us to undertake a bioinformatic search for new O2‐, PLP‐dependent enzymes. We identified one widely distributed enzyme, which we named RohP. Through in vitro biochemical studies we characterized the activity of RohP and found that it uses PLP and O2 to catalyze the hydroxylation of an sp3‐hybridized carbon in l‐arginine, giving 4‐hydroxy‐2‐ketoarginine, along with stoichiometric conversion of O2 to H2O2. Surprisingly, we found that the hydroxyl group in the 4‐hydroxy‐2‐ketoarginine product is derived from water, raising questions about the mechanism of RohP. To provide insight into the mechanism behind this hydroxylation, we used X‐ray crystallography to obtain four ~1.5 Å resolution snapshots interpreted to represent RohP at different stages of its catalytic cycle: the holo enzyme, two different PLP‐bound intermediates, and the enzyme in complex with (S)‐4‐hydroxy‐2‐ketoarginine. These structures reveal that the dynamic N‐terminus of RohP, which is required for catalysis, becomes ordered upon substrate binding, sealing off the active site from the bulk solvent. Concurrently, several conformational changes of residues in the active site help to properly orient the substrate for catalysis. Through our structural work we also identify a conserved histidine, which we demonstrate is essential for the later steps in catalysis, and we suggest that this residue may be key to catalyzing a stereospecific alkene hydration. Collectively, our biochemical and X‐ray crystallographic investigation provides insights into how O2‐, PLP‐dependent oxidases can catalyze the challenging hydroxylation of an unactivated sp3‐hybridized carbon in l‐arginine and sets the stage for further mechanistic studies on this exciting group of enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.