During the biosynthesis of the peptide antibiotic rufomycin, the cytochrome P450 (P450) enzyme 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 homologue 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 explain how sequence differences in RufO and its homologues, along with the structure of the pentapeptide substrate, facilitate aromatic nitration of tyrosine by RufO.
A. M. Kirk, Jemma Gullick, Yong-Wei Zhao et al.· Journal of the American Chem...· 0 citations
Staphylococcus aureus strains have emerged with resistance mechanisms that reduce the efficacy of last resort antibiotics and evade the immune system. One strategy to combat antimicrobial resistance is to modulate host immunity to eliminate infections more effectively. This has led to the development of immunotherapeutics consisting of vancomycin conjugated to formyl peptides (fPeps), with vancomycin targeting the cell wall and the fPeps engaging host innate immunity. Here, we used flow cytometry to quantify the binding of vancomycin=fPep conjugates to S. aureus clinical isolates. This revealed reduced binding of vancomycin=fPeps compared to vancomycin alone and quantified the interaction between the conjugates and the bacterial cell surface, which is important to quantify to then control the chemotactic gradient established by the fPep cargo. The direct antimicrobial activity of these conjugates was also reduced when compared to vancomycin, reflecting the reduced binding of these conjugates to S. aureus. This flow cytometry method allows quantification of vancomycin=fPep binding to bacteria and will assist in future studies to understand how attached fPeps and other immune signalling cargoes can stimulate innate immune cell activation leading to bacterial phagocytosis.
Winfrey P Y Hoo, Jemma Gullick, Ryan Leung et al.· ChemBioChem· 0 citations
This study repurposed a machine learning algorithm to comprehensively chart the biosynthetic space of the biarylitides, including variation of precursor motifs, P450, and additional modifying enzymes, which yielded 277 biarylitide biosynthetic gene clusters (BGCs).
Leo Padva, Jemma Gullick, Friederike Biermann et al.· JACS Au· 1 citation
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