The biosynthesis of methanobactin

Abstract

Metal homeostasis poses a major challenge to microbes, which must acquire scarce elements for core metabolic processes. Methanobactin, an extensively modified copper-chelating peptide, was one of the earliest natural products shown to enable microbial acquisition of a metal other than iron. We describe the core biosynthetic machinery responsible for the characteristic posttranslational modifications that grant methanobactin its specificity and affinity for copper. A heterodimer comprising MbnB, a DUF692 family iron enzyme, and MbnC, a protein from a previously unknown family, performs a dioxygen-dependent four-electron oxidation of the precursor peptide (MbnA) to install an oxazolone and an adjacent thioamide, the characteristic methanobactin bidentate copper ligands. MbnB and MbnC homologs are encoded together and separately in many bacterial genomes, suggesting functions beyond their roles in methanobactin biosynthesis.

Chalkophores

“…Importantly, this mechanism of heterocycle formation appears to apply to MbnA/MbnBC pairs from all five operon groups, although Group V is sufficiently divergent so that its enzyme and substrate are not compatible with the other four groups. An alternative mechanism involving a catalytic thiol and several biosynthetically unusual steps has been suggested (62), but even apart from the lack of conserved cysteines in MbnB and MbnC, this model does not fit the biochemical data, which support an oxygen- and iron-dependent mechanism (92) (Figure 2 b ). …”

“…Instead, recent work indicates that MbnB and MbnC form an entirely novel heterodimeric iron-containing enzyme (MbnBC) that carries out a four-electron oxidation on the MbnA substrate, forming an oxazolone and thioamide moiety from a cysteine in a single concerted rearrangement (92). The iron active site is located in the MbnB subunit, and some ambiguity remains regarding the composition and oxidation state of the active cofactor, with coupled di- and trinuclear iron centers as possibilities.…”

“…However, the observed mass is consistent with acid-catalyzed oxazolone hydrolysis (45), which in combination with the predicted lack of an N-terminal transamination reaction, provides a potential explanation for the missing oxazolone ring. Indeed, addition of copper during purification stabilizes the Mbn intermediate and prevents oxazolone degradation (92); the role of MbnN is thus predicted to be catalyzing a simple transamination reaction on this intermediate. A biosynthetic role has also been proposed for the diheme cytochrome c peroxidase MbnH and its partner protein MbnP (62); however, mbnPH genes are commonly associated with genes related to CuMbn uptake (25), and while the Ms. sp.…”

“…LW4 Mbn operon lacks mbnPH , its Mbn contains oxazolone/thioamide moieties (75). On the basis of in vitro experiments with MbnBC, it does not appear that there is a role for either MbnN or MbnPH in oxazolone/thioamide biosynthesis (92). …”

“…One gene conspicuously absent in all Mbn operons is a protease responsible for leader peptide cleavage. Whether MbnB or MbnC is capable of catalyzing MbnA leader peptide cleavage is unclear, although this proteolytic step has not been observed in vitro (92), or whether an unknown process is responsible.…”