MbnC is not required for the formation of the N-terminal oxazolone in the methanobactin from Methylosinus trichosporium 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 Δ mbn C mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry and solution NMR spectroscopy. MB-OB3b from Δ mbn C 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.

The Ethylmalonyl-CoA Pathway for Methane-based Biorefineries: A Case Study of Using Methylosinus trichosporium OB3b, an Alpha-proteobacterial Methanotroph, for Producing 2-Hydroxyisobutyric Acid and 1,3-Butanediol from Methane

The ethylmalonyl-CoA pathway is one of three known anaplerotic pathways that replenish tricarboxylic acid cycle intermediates and plays a major role in the carbon metabolism of many alpha-proteobacteria including Methylosinus...

Genome-Scale Metabolic Model Reconstruction and in Silico Investigations of Methane Metabolism in Methylosinus trichosporium OB3b

Methylosinus trichosporium OB3b is an obligate aerobic methane-utilizing alpha-proteobacterium. Since its isolation, M. trichosporium OB3b has been established as a model organism to study methane metabolism in type II methanotrophs. M. trichosporium OB3b utilizes soluble and particulate methane monooxygenase (sMMO and pMMO respectively) for methane oxidation. While the source of electrons is known for sMMO, there is less consensus regarding electron donor to pMMO. To investigate this and other questions regarding methane metabolism, the genome-scale metabolic model for M. trichosporium OB3b (model ID: iMsOB3b) was reconstructed. The model accurately predicted oxygen: methane molar uptake ratios and specific growth rates on nitrate-supplemented medium with methane as carbon and energy source. The redox-arm mechanism which links methane oxidation with complex I of electron transport chain has been found to be the most optimal mode of electron transfer. The model was also qualitatively validated on ammonium-supplemented medium indicating its potential to accurately predict methane metabolism in different environmental conditions. Finally, in silico investigations regarding flux distribution in central carbon metabolism of M. trichosporium OB3b were performed. Overall, iMsOB3b can be used as an organism-specific knowledgebase and a platform for hypothesis-driven theoretical investigations of methane metabolism.