The biogenic methanobactin is an effective chelator for copper in a rat model for Wilson disease.

Copper is an essential redox-active metal ion which in excess becomes toxic due to the formation of reactive oxygen species. In Wilson disease the elevated copper level in liver leads to chronic oxidative stress and subsequent hepatitis. This study was designed to evaluate the copper chelating efficiency of the bacterial methanobactin (MB) in a rat model for Wilson disease. Methanobactin is a small peptide produced by the methanotrophic bacterium Methylosinus trichosporium OB3b and has an extremely high affinity for copper. Methanobactin treatment of the rats was started at high liver copper and serum aspartate aminotransferase (AST) levels. Two dosing schedules with either 6 or 13 intraperitoneal doses of 200mg methanobactin per kg body weight were applied. Methanobactin treatment led to a return of serum AST values to basal levels and a normalization of liver histopathology. Concomitantly, copper levels declined to 45% and 24% of untreated animals after 6 and 13 doses, respectively. Intravenous application of methanobactin led to a prompt release of copper from liver into bile and the copper was shown to be associated with methanobactin. In vitro experiments with liver cytosol high in copper metallothionein demonstrated that methanobactin removes copper from metallothionein confirming the potent copper chelating activity of methanobactin

Oxygen generation via water splitting by a novel biogenic metal ion binding compound.

Methanobactins (MBs) are small (<1,300 Da) post-translationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some reduced after binding, e.g., Cu2+ reduced to Cu+ Other metal ions, however, are bound but not reduced, e.g., K+ The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H218O, we show that when MB from Methylocystis sp strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au3+, Cu2, and Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed either in presence of MB-SB2 or MB-OB3b alone, gold alone, copper alone, silver alone or when K+ or Mo2+ was incubated with MB-SB2.In contrast to MB-OB3b, MB-SB2 binds Fe3+ with an N2S2 coordination and will also reduce Fe3+ to Fe2+ Iron reduction was also found to be coupled to oxidation of 2H2O and generation of O2 MB-SB2 will also couple Hg2+, Ni2+ and Co2+ reduction to the oxidation of 2H2O and generation of O2, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions.To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate O2 generation from metal ion reduction by MB-OB3b can support methane oxidation.IMPORTANCEThe discovery that MB will couple the oxidation of H2O to metal ion reduction and the release of O2 suggests that methanotrophs expressing MB may be able to maintain their activity in hypoxic/anoxic conditions through "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest, but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere

MbnC is not required for the formation of the N-terminal oxazolone in the methanobactin from <em>Methylosinus trichosporium</em> OB3b.

Methanobactins (MBs) are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The post-translational modification that define MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b, mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons, and in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry and solution NMR spectroscopy. MB-OB3b from ΔmbnC was missing the C-terminal Met and also found to contain a Pro and a Cys in place of the pyrrolidiny-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals, nanoparticle formation, as well as for the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for post-translational modification(s) of the two oxazolone groups are not identical

Draft genome sequence of the volcano-inhabiting thermoacidophilic methanotroph methylacidiphilum fumariolicum strain solv

Contains fulltext : 93817.pdf (author's version ) (Open Access

Evidence for methanobactin "Theft" and novel chalkophore production in methanotrophs: Impact on methanotrophic-mediated methylmercury degradation.

Aerobic methanotrophy is strongly controlled by copper, and methanotrophs are known to use different mechanisms for copper uptake. Some methanotrophs secrete a modified polypeptide-methanobactin-while others utilize a surface-bound protein (MopE) and a secreted form of it (MopE*) for copper collection. As different methanotrophs have different means of sequestering copper, competition for copper significantly impacts methanotrophic activity. Herein, we show that Methylomicrobium album BG8, Methylocystis sp. strain Rockwell, and Methylococcus capsulatus Bath, all lacking genes for methanobactin biosynthesis, are not limited for copper by multiple forms of methanobactin. Interestingly, Mm. album BG8 and Methylocystis sp. strain Rockwell were found to have genes similar to mbnT that encodes for a TonB-dependent transporter required for methanobactin uptake. Data indicate that these methanotrophs "steal" methanobactin and such "theft" enhances the ability of these strains to degrade methylmercury, a potent neurotoxin. Further, when mbnT was deleted in Mm. album BG8, methylmercury degradation in the presence of methanobactin was indistinguishable from when MB was not added. Mc. capsulatus Bath lacks anything similar to mbnT and was unable to degrade methylmercury either in the presence or absence of methanobactin. Rather, Mc. capsulatus Bath appears to rely on MopE/MopE* for copper collection. Finally, not only does Mm. album BG8 steal methanobactin, it synthesizes a novel chalkophore, suggesting that some methanotrophs utilize both competition and cheating strategies for copper collection. Through a better understanding of these strategies, methanotrophic communities may be more effectively manipulated to reduce methane emissions and also enhance mercury detoxification in situ

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

In Wilson disease (WD), functional loss of ATPase copper-transporting &beta; (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD