Provision of a Redox Donor to Oxy-Ferrous PrnB Is Required for Pyrrolnitrin Synthesis

PrnB, a long-standing member of the recently reclassified heme-dependent aromatic oxygenase (HDAO) superfamily of histidine-ligated heme enzymes, catalyzes the conversion of 7-chloro-l-tryptophan (7-Cl-Trp) to monodechloroaminopyrrolnitrin (MCAP). This unique ring rearrangement is an essential step in the biosynthesis of the broad-spectrum antifungal pyrrolnitrin. The conversion of 7-Cl-Trp to MCAP by PrnB differs from other HDAOs, which typically affect the mono- or dioxygenation of aromatic substrates. However, the molecular basis for this transformation has remained enigmatic due to the inability to reconstitute enzymatic activity in vitro. Transient kinetic approaches reveal that the sequential binding of 7-Cl-Trp and dioxygen results in the formation of a long-lived oxy-ferrous complex. Despite similarity to intermediates found in other HDAOs, the PrnB-oxy ternary species does not react with bound 7-Cl-Trp. However, the provision of a surrogate redox delivery system supports turnover, and single-turnover studies reveal that activation of the oxy-complex is required. Together, these studies reveal the molecular basis for functional expansion of the HDAO structural framework through alteration of the strategy of oxygen activation.