Pyrrolnitrin, a potent antifungal compound originally discovered in Pseudomonas strains, is biosynthesized through a secondary metabolic pathway involving four key enzymes. Central to this process is PrnB, a heme enzyme that catalyzes the complex transformation of 7-Cl-L-tryptophan. Despite its structural similarity to indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) and its classification within the histidine-ligated heme-dependent aromatic oxygenase (HDAO) superfamily, PrnB has remained relatively unexplored due to challenges in reconstituting its in vitro activity. In this work, we investigated the interactions of PrnB from different strains with its substrates, substrate analogs, and small molecules using various biophysical and biochemical techniques. Our spectroscopic data reveal that the substrate amino group directly coordinates with the heme in both oxidized and reduced enzyme forms. This binding conformation was further confirmed by X-ray crystallography of enzyme-ligand binary complexes. The amine ligation inhibits H2O2 and CN- from interacting with the ferric heme but does not notably impact •NO binding or O2 activation by the ferrous heme. Stopped-flow spectroscopy showed the formation of heme-based oxidants similar to those reported in IDO and TDO when PrnB was exposed to H2O2 or O2. However, these intermediates lacked catalytic activity, and PrnB was inactive when coupled with common redox systems under various conditions. This suggests that PrnB operates through a catalytic mechanism distinct from other HDAOs and most heme enzymes. Our study provides new insights into ligand binding and small-molecule activation mechanisms of PrnB, highlighting its unique functionality and distinguishing it from existing paradigms in heme catalysis.
Keywords: crystal structure; heme-based oxidants; heme-dependent catalysis; pyrrolnitrin biosynthesis; spectroscopic characterization; tryptophan oxidation.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.