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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1209496810
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu12094968102021-08-03T05:53:34Z Methyltransferases and Corrinoid Binding Proteins Involved in Methylamine Dependent Methanogenesis in Methanosarcina Barkeri Ferguson, Donald Jay, Jr. <p><i>Methanosarcina barkeri</i> is a methanogen capable of producing methane from a wide range of substrates including carbon dioxide with molecular hydrogen, acetate, methylated sulfur compounds, methanol, and the methylamines. In all known pathways of methanogenesis, the penultimate step involves the methylation of 2-mercaptoethanesulfonate (CoM). At the onset of this investigation, the pathways of CoM methylation from CO<sub>2</sub>/H<sub>2</sub>, acetate, and methanol were largely understood, however, methylamine dependent CoM methylation was still uncharacterized. Therefore, this study was undertaken to identify the role of the methylcorrinoid:CoM methyltransferase, MtbA, in methylamine dependent CoM methylation, isolate and characterize the enzymes involved in the initial steps of CoM methylation from trimethylamine (TMA) and dimethylamine (DMA), and to reconstitute the pathways of CoM methylation from TMA and DMA <i>in vitro</i>.</p><p>An immunochemical approach was employed as a test for the functional activities of the methylcorrinoid:CoM methyltransferases, MtbA and MtaA, in the metabolic pathways of methane formation from monomethylamine (MMA), DMA, and TMA. Specific removal and subsequent addition of MtbA from extracts of TMA grown cells demonstrated the involvement of MtbA in the pathways of CoM methylation from TMA, DMA, and MMA. It was shown that MtbA functions as the exclusive methylcorrinoid:CoM methyltransferase in the MMA:CoM and DMA:CoM methyl transfer pathways. However, both MtbA and MtaA were shown to function in the TMA:CoM methyl transfer pathway.</p><p>Reconstitution of the TMA:CoM methyl transfer pathway was achieved with three purified polypeptides: MttB, MttC, and MtbA. Two of these polypeptides copurified as the MttBC complex which stimulated TMA:CoM methyl transfer activity in extracts. MttB is a 280 kDa protein composed of 52 kDa subunits and MttC is a 26 kDa protein which can exist as a monomer or in complex with MttB. The 26 kDa MttC was shown to bind 1.1 mol corrinoid per mol polypeptide whereas MttB bound no apparent prosthetic group. It was found that either methylcorrinoid:CoM methyltransferase could interact with MttC to carry out TMA:CoM methyl transfer although MttC showed a greater affinity for MtbA than for MtaA.</p><p>DMA dependent CoM methylation was reconstituted <i>in vitro</i> for the first time using only highly purified proteins. These proteins included the previously unidentified corrinoid protein, MtbC, which copurified with MtbA, and a DMA:corrinoid methyltransferase MtbBl. MtbC was shown to bind 1 mol corrinoid per mol polypeptide whereas MtbBl bound no apparent prosthetic group. A molar ratio of MtbC to MtbBl of 1 was found to be optimal for activity. Other than the ability of MtbBl to methylate free cob(I)alamin with DMA at a low rate, MtbC and MtbBl showed a high degree of specificity for one another and for DMA as the substrate.</p> 2000 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1209496810 http://rave.ohiolink.edu/etdc/view?acc_num=osu1209496810 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
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NDLTD
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English
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NDLTD
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| author |
Ferguson, Donald Jay, Jr.
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Ferguson, Donald Jay, Jr.
Methyltransferases and Corrinoid Binding Proteins Involved in Methylamine Dependent Methanogenesis in Methanosarcina Barkeri
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| author_facet |
Ferguson, Donald Jay, Jr.
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| author_sort |
Ferguson, Donald Jay, Jr.
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| title |
Methyltransferases and Corrinoid Binding Proteins Involved in Methylamine Dependent Methanogenesis in Methanosarcina Barkeri
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| title_short |
Methyltransferases and Corrinoid Binding Proteins Involved in Methylamine Dependent Methanogenesis in Methanosarcina Barkeri
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| title_full |
Methyltransferases and Corrinoid Binding Proteins Involved in Methylamine Dependent Methanogenesis in Methanosarcina Barkeri
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| title_fullStr |
Methyltransferases and Corrinoid Binding Proteins Involved in Methylamine Dependent Methanogenesis in Methanosarcina Barkeri
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| title_full_unstemmed |
Methyltransferases and Corrinoid Binding Proteins Involved in Methylamine Dependent Methanogenesis in Methanosarcina Barkeri
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| title_sort |
methyltransferases and corrinoid binding proteins involved in methylamine dependent methanogenesis in methanosarcina barkeri
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| publisher |
The Ohio State University / OhioLINK
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| publishDate |
2000
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| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1209496810
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| work_keys_str_mv |
AT fergusondonaldjayjr methyltransferasesandcorrinoidbindingproteinsinvolvedinmethylaminedependentmethanogenesisinmethanosarcinabarkeri
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| _version_ |
1719427273714565120
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