Degree Type

Dissertation

Date of Award

2017

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Alan A. DiSpirito

Abstract

Methanobactins (mbs) are low molecular mass (< 1300 Da) modified peptides

secreted by many methanotrophs or methane oxidizing bacteria to sequester copper from the

environment. To date, methanobactin has been structurally characterized from six

methanotrophs and can be divided into two groups. Group I methanobactins are represented

by methanobactins from Methylosinus trichosporium OB3b and Methylosinus sp. LW4. This

group is characterized by the presence of two oxazolone rings with adjacent thioamide

groups. Two nitrogens from the oxazolone rings and two sulfurs from the thioamide groups

come together to form a copper coordination site in distorted tertrahedral geometry. This

group of methanobactin also has two cysteines in the mature protein that form a stable

disulfide bond. The second group is composed of methanobactins from four different

Methylocystis species. This group of methanobactins has a hairpin like structure following

copper binding with sulfate group attached to serine or threonine and is structurally more

dynamic than group I methanobactins. This group is also characterized by the presence of a

C-terminal oxazolone ring with an associated thioamide and either an N-terminal

imidazolone or pyrazinedione group and an associated thioamide.

The first part of this dissertation is focused on the study of methanobactin binding to various non-copper metals such as mercury and gold and the role of that binding of noncopper

metals has on the physiology of methanotrophs. The second part of this dissertation is

focused on understanding the post-translational modifications required for methanobactin.

Until recently, the biosynthesis was assumed to be via a non-ribosomal peptide synthase or

polyketide synthase. However, during the course of my dissertation, we determined that

methanobactin is indeed produced ribosomally and post-translationally modified. I show in

this dissertation that TonB-dependent transporter gene in the methanobactin gene cluster is

involved in the uptake of copper-bound methanobactin. In addition, we demonstrate that

mbnN is involved in the deamination of the N-terminal oxazolone ring, a post-translational

modification required in the formation of the N-terminal oxazolone ring in the methanobactin from Methylosinus trichosporium OB3b.

The results and methods used in this research would further help determine the role of

other genes involved in biosynthesis of methanobactin and bioengineer methanobactin for

various human and animal health purposes. In light of recent evidence of methanobactin

being effective chelator of excess copper in Wilson’s disease in rat models, understanding the

biosynthesis of methanobactin has become more important.

DOI

https://doi.org/10.31274/etd-180810-4884

Copyright Owner

Bipin Sharma Baral

Language

en

File Format

application/pdf

File Size

199 pages

Included in

Biochemistry Commons

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