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 posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. B...

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Bibliographic Details
Published in:Applied and environmental microbiology 2022-01, Vol.88 (2), p.e0184121
Main Authors: Dershwitz, Philip, Gu, Wenyu, Roche, Julien, Kang-Yun, Christina S, Semrau, Jeremy D, Bobik, Thomas A, Fulton, Bruce, Zischka, Hans, DiSpirito, Alan A
Format: Article
Language:English
Subjects:
Absorption spectroscopy
Bacteriology
Copper - metabolism
Gene deletion
Genes
Genetics and Molecular Biology
Imidazoles - metabolism
Mass spectrometry
Mass spectroscopy
Methanotrophic bacteria
Methylosinus trichosporium
Mutants
Mutation
NMR
NMR spectroscopy
Nuclear magnetic resonance
Oligopeptides - metabolism
Operons
Oxazolone
Oxazolone - metabolism
Oxygenases - metabolism
Peptides
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Summary:Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines 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 is , which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, , is found in all MB operons and, in conjunction with , is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of , a deletion mutation was constructed in OB3b, and the MB produced from the Δ mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the Δ mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-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. A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as 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 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 OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.
ISSN:0099-2240
1098-5336
DOI:10.1128/AEM.01841-21