Functional and Structural Characterization of a Methanobactin Transporter

Project: Research project

Project Details

Description

Natural products have been identified in numerous organisms and have been used as pharmacological agents, secondary metabolites, and chemical agents, among others. A common class of natural products is those that bind metals and are taken into cells to fulfill the cell’s metal needs. The vast
majority of these natural products bind iron and are referred to as siderophores (iron “carriers”). Similar molecules that bind copper rather than iron have been discovered recently. These molecules were initially
isolated from the growth media of methanotrophic bacteria (bacteria that metabolize methane), which have an elevated copper requirement. These “chalkophores” (copper “carriers”) have been termed methanobactins (Mbns). Mbns are secreted when methanotrophic bacteria are grown under conditions of limiting copper. Mbns acquire copper from the environment and are taken up into the cell. Mbns have a high affinity for copper, and this has sparked interest in their potential use for the treatment of diseases of toxic copper overload. The mechanism by which copper-loaded Mbn is taken up into methanotrophic bacteria is the focus of this research. Uptake of Mbn has been demonstrated in the methanotrophic bacteria Methylosinus trichosporium (Mt) OB3b, and proceeds via an energy-dependent process using the TonB complex of proteins. The TonB complex transduces energy from the inner membrane to the outer membrane, and
interacts with TonB-dependent transporters (TBDTs) in the outer membrane to facilitate the internalization of siderophores and other secondary metabolites that are too large to enter the cell via diffusion. There are 45 TBDTs in the genome of Mt OB3b. The particular TBDT protein responsible for uptake of Mbn in Mt OB3b will be identified and its mechanism investigated as described in the following specific aims: 1) in vivo assays to demonstrate that the lone TBDT protein indentified in the mbn operon facilitates Mbn uptake in a heterologous host; 2) in vitro biochemical assays with the protein to determine the thermodynamics of binding Mbn and the specificity of the protein for Mbn; and X-ray crystallographic characterization of the protein to determine the structural basis for Mbn transport.
StatusFinished
Effective start/end date9/1/158/31/18

Funding

  • Burroughs Wellcome Fund (1015098)

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