A Mechanism for Aromatic Nitration Catalyzed by Cytochrome P450 Enzymes from Rufomycin Biosynthesis

During the biosynthesis of the peptide antibiotic rufomycin, the cytochrome P450 (P450) RufO catalyzes the aromatic nitration of a tyrosine residue in a ribosomal pentapeptide that serves as a precursor for nonribosomal peptide synthesis. To understand the mechanism of this unusual P450-mediated reaction, a series of pentapeptides were tested as substrates alongside molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM, ONIOM approach) calculations. A new substrate bound crystal structure of the homolog NsRufO was also obtained. These experiments revealed the intimate and necessary involvement of a histidine residue within the pentapeptide substrate of RufO in supporting effective nitration, with the protonation state of the intermediate Compound II proposed to influence the production of nitrated tyrosine over unwanted nitrate formation. These findings provide key insights into the mechanism of P450-mediated nitration that explains how sequence differences in RufO and its analogues, along with the structure of the pentapeptide substrate, facilitate aromatic nitration of tyrosine by RufO.