NMR, Mass Spectrometry and Chemical Evidence Reveal a Different Chemical Structure for Methanobactin That Contains Oxazolone Rings

Abstract

Methanobactin (mb) is a small copper-binding peptide produced by methanotrophic bacteria and is intimately involved in both their copper metabolism and their role in the global carbon cycle. The structure for methanobactin comprises seven amino acids plus two chromophoric residues that appear unique to methanobactin. In a previously published structure, both chromophoric residues contain a thiocarbonyl attached to a hydroxyimidazolate ring. In addition, one is attached to a pyrrolidine ring, while the other to an isopropyl ester. A published X-ray determined structure for methanobactin shows these two chromophoric groups forming an N2S2 binding site for a single Cu(I) ion with distorted tetrahedral geometry. In this report we show that NMR, mass spectrometry, and chemical data, reveal a chemical structure that is significantly different than the previously published one. Specifically, the 1H and 13C NMR assignments are inconsistent with an N-terminal isopropyl ester and point instead to a 3-methylbutanoyl group. Our data also indicate that oxazolone rings instead of hydroxyimidazolate rings form the core of the two chromophoric residues. Because these rings are directly involved in the binding of Cu(I) and other metals by methanobactin, and are likely involved in the many chemical activities displayed by methanobactin, their correct identity is central to developing an accurate and detailed understanding of methanobactin’s many chemical and biological roles. For example, the oxazolone rings make methanobactin structurally more similar to other bacterially produced bactins and siderophores and suggest pathways for its biosynthesis.

Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

“…Do all forms of methanobactin exhibit the same ability to bind gold or other group A metals? Given the significant similarity between the known forms of methanobactin (23)(24)(25)(26), it is plausible to presume that they would all bind gold to some degree, but the resultant impact on MMO expression is less clear. For example, some nonswitchover methanotrophs, i.e., those that can express only pMMO, are known to produce methanobactin (24,25).…”

“…For the sequestration of copper, many methanotrophs have been found to synthesize different chalkophores for copper uptake, akin to siderophores used for iron uptake. These chalkophores, collectively called methanobactin, are small modified polypeptides (Ͻ1,200 Da) with two heterocyclic rings (one of which is an oxazolone ring and the other of which is either an oxazolone, imidazolone, or pyrazinedione ring) and associated enethiol groups (23)(24)(25)(26). Copper is bound by methanobactin with very high affinity (reported affinities range from 10 20 to 10 34 M Ϫ1 [24,[27][28][29][30]) using nitrogen from both heterocyclic rings and the sulfur of the enethiol groups.…”

Liver mitochondrial membrane crosslinking and destruction in a rat model of Wilson disease

“…*P < 0.05, ***P < 0.001 versus the preceding age group; † P < 0.001 versus control; t test. equimolar doses of the copper chelator bathocuproine disulfonic acid (BCDS) (Figure 5B) or by the peptide methanobactin (Supplemental Figure 4B), which has a high copper-binding affinity (37).…”

Methanobactin reverses acute liver failure in a rat model of Wilson disease

“…One-way ANOVA with Tukey's multiple comparisons test. ****P < 0.0001 versus control; comprises 7 aa plus 2 chromophoric residues (termed OxaA and OxaB) forming an N 2 S 2 binding site for a single copper ion (26). Methanotrophic proteobacteria have a high copper requirement for their copper-dependent methane oxidase (27,28) and excrete MB to sequester extracellular copper (25,27,28).…”