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An Aminotransferase Is Responsible for the Deamination of the N-Terminal Leucine and Required for Formation of Oxazolone Ring A in Methanobactin of Methylosinus trichosporium OB3b
- Source :
- Applied and Environmental Microbiology. 83
- Publication Year :
- 2017
- Publisher :
- American Society for Microbiology, 2017.
-
Abstract
- 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-Ψ) 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.
- Subjects :
- 0301 basic medicine
Methane monooxygenase
Genetic Vectors
030106 microbiology
Glycine
Deamination
Gene Expression
Genetics and Molecular Biology
Applied Microbiology and Biotechnology
Oxazolone
03 medical and health sciences
chemistry.chemical_compound
Methionine
Biosynthesis
Leucine
Gene cluster
Transaminases
Ecology
biology
Chemistry
Imidazoles
Wild type
Gene Expression Regulation, Bacterial
Methanobactin
Methylosinus trichosporium
Thioamides
030104 developmental biology
Biochemistry
Multigene Family
biology.protein
Oligopeptides
Protein Processing, Post-Translational
Copper
Gene Deletion
Food Science
Biotechnology
Subjects
Details
- ISSN :
- 10985336 and 00992240
- Volume :
- 83
- Database :
- OpenAIRE
- Journal :
- Applied and Environmental Microbiology
- Accession number :
- edsair.doi.dedup.....c2028f852ca8e87d06113fbe71986336