Methanobactin biosynthesis and redox activity

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2022-12
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Dershwitz, Philip
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DiSpirito, Alan
Bobik, Thomas
Honzatko, Richard
Roche, Julien
Phillips, Gregory
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Biochem, Biophysics, and Molecular Biology
Abstract
Methanobactins are low-molecular-mass (<1,300 Da), ribosomally synthesized and posttranslationally modified copper-binding peptides excreted by some methanotrophs as the extracellular component of a copper acquisition system. Structurally, methanobactins are characterized by the presence of a C-terminal oxazolone group with a C2-associated thioamide and by the presence of an N-terminal oxazolone, imidazolone or pyrazinedione group with an associated thioamide. Some methanobactins also contain a sulfate group in-place of the hydroxyl group on a Tyr adjacent to the C-terminal oxazolone group. The two nitrogens on the rings and the two thioamide sulfurs are the four atoms that coordinate with copper and are the source of methanobactins’ copper-binding ability. Methanobactins with two oxazolone groups are classified as Group I, while those with only one are classified as Group II. Group I methanobactins bind copper to form a stable tetrahedral structure, while Group II methanobactins form a more dynamic hairpin structure. These structural differences give rise to the two groups’ physical and functional differences. The first part of this dissertation focuses on a methanobactin-catalyzed redox reaction in which O2 is generated from H2O, the difference in activity between different methanobactins, and the functional and ecological implications of this reaction. Specifically, in methanotrophs that produce methanobactins, their ability to generate their own oxygen could allow them to perform aerobic methane oxidation in a range of environments in which that reaction would otherwise not be possible. The second part of this dissertation focuses on the biosynthesis of methanobactin from Methylosinus trichosporium OB3b. As ribosomally synthesized and posttranslationally modified peptides, methanobactins have an entire operon dedicated to the transport, chaperoning and modification of the proto-methanobactin gene product from the mbnA gene. Several of the genes in this operon were and are unannotated, including mbnC. To that end a ΔmbnC knockout mutant was created and the structure of its resulting methanobactin was solved by metal-free NMR in order to determine that mbnC is not required for the formation of the N-terminal oxazolone the methanobactin from M. trichosporium OB3b. Finally, to elucidate the function of mbnF from Methylocystis strain SB2, the gene product was isolated, a crystal structure of the protein was solved, and preliminary enzymology was performed. From this work it was determined that mbnF encodes a NADPH-dependent flavin monooxygenase. The results and methods in this research advance our understanding of the function and biosynthesis of methanobactin. In light of methanobactins’ utility as a copper chelator for the treatment of Wilson’s disease in a rat model, and methanotrophs’ critical importance in the global carbon cycle, a better understanding of methanobactins’ biosynthesis and catalytic activity could have far-reaching consequences for society.
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