Dataset Information

Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b.

ABSTRACT:

Background

The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as well as with elemental analysis and ESI-MS.

Results

The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/N ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances.

Conclusion

Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin.

SUBMITTER: Pesch ML

PROVIDER: S-EPMC3044663 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Publications

Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b.

Pesch Marie-Laure ML Christl Iso I Barmettler Kurt K Kraemer Stephan M SM Kretzschmar Ruben R

Geochemical transactions 20110207

<h4>Background</h4>The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and cha ...[more]

PMID: 21299876

Similar Datasets

Project description:Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper-regulating expression of alternative forms of methane monooxygenase. A copper-binding compound, or chalkophore, called methanobactin plays a key role in copper uptake in methanotrophs. Methanobactin is a ribosomally synthesized and posttranslationally modified peptide (RiPP) with two heterocyclic rings with an associated thioamide for each ring, formed from X-Cys dipeptide sequences that bind copper. The gene coding for the precursor polypeptide of methanobactin, mbnA, is part of a gene cluster, but the role of other genes in methanobactin biosynthesis is unclear. To begin to elucidate the function of these genes, we constructed an unmarked deletion of mbnABCMN in Methylosinus trichosporium OB3b and then homologously expressed mbnABCM using a broad-host-range cloning vector to determine the function of mbnN, annotated as coding for an aminotransferase. Methanobactin produced by this strain was found to be substantially different from wild-type methanobactin in that the C-terminal methionine was missing and only one of the two oxazolone rings was formed. Rather, in place of the N-terminal 3-methylbutanoyl-oxazolone-thioamide group, a leucine and a thioamide-containing glycine (Gly-?) were found, indicating that MbnN is used for deamination of the N-terminal leucine of methanobactin and that this posttranslational modification is critical for closure of the N-terminal oxazolone ring in M. trichosporium OB3b. These studies provide new insights into methanobactin biosynthesis and also provide a platform for understanding the function of other genes in the methanobactin gene cluster. IMPORTANCE:Methanotrophs, microbes that play a critical role in the carbon cycle, are influenced by copper, with gene expression and enzyme activity changing as copper levels change. Methanotrophs produce a copper-binding compound, or chalkophore, called methanobactin for copper uptake, and methanobactin plays a key role in controlling methanotrophic activity. Methanobactin has also been shown to be effective in the treatment of Wilson disease, an autosomal recessive disorder where the human body cannot correctly assimilate copper. It is important to characterize the methanobactin biosynthesis pathway to understand how methanotrophs respond to their environment as well as to optimize the use of methanobactin for the treatment of copper-related diseases such as Wilson disease. Here we show that mbnN, encoding an aminotransferase, is involved in the deamination of the N-terminal leucine and necessary for the formation of one but not both of the heterocyclic rings in methanobactin that are responsible for copper binding.

Dataset Information

Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b.

Background

Results

Conclusion

Publications

Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b.

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