Search for: All records

The iron-containing heterodimeric MbnBC enzyme complex plays a central role in the biosynthesis of methanobactins (Mbns), ribosomally synthesized, posttranslationally modified natural products that bind copper with high affinity. MbnBC catalyzes a four-electron oxidation of a cysteine residue in its precursor-peptide substrate, MbnA, to an oxazolone ring and an adjacent thioamide group. Initial studies of MbnBC indicated the presence of both diiron and triiron species, complicating identification of the catalytically active species. Here, we present evidence through activity assays combined with electron paramagnetic resonance (EPR) and Mössbauer spectroscopic analysis that the active species is a mixed-valent, antiferromagnetically coupled Fe(II)Fe(III) center. Consistent with this assignment, heterologous expression of the MbnBC complex in culture medium containing less iron yielded purified protein with less bound iron but greater activity in vitro. The maximally activated MbnBC prepared in this manner could modify both cysteine residues in MbnA, in contrast to prior findings that only the first cysteine could be processed. Site-directed mutagenesis and multiple crystal structures clearly identify the two essential Fe ions in the active cluster as well as the location of the previously detected third Fe site. Moreover, structural modeling indicates a role for MbnC in recognition of the MbnA leader peptide. These results add a biosynthetic oxidative rearrangement reaction to the repertoire of nonheme diiron enzymes and provide a foundation for elucidating the MbnBC mechanism.