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 <i>in vitro</i>. 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.