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 Title: Characterization of Enzymes of the Methylcoenzyme M Reductase System From Methanobacterium Thermoautotrophicum and Identification of a Factor Required for Growth of Methanomicrobium Mobile Author(s): Kuhner, Carla Heidi Doctoral Committee Chair(s): Wolfe, R.S. Department / Program: Microbiology Discipline: Microbiology Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Biology, Microbiology Abstract: The reduction of 2-(methylthio) ethanesulfonate (CH$\sb3$-S-coenzyme M, CH$\sb3$-S-CoM) by methanogenic archaea is catalyzed by a methylcoenzyme M reductase (MR). In Methanobacterium thermoautotrophicum strain $\Delta$H, this reaction has been shown to require minimally two additional protein components (A2 and A3a), as well as ATP and a second reductant. Although component A2 has been purified to homogeneity, proteins comprising component A3a have only been partially resolved, and the functions of these components remain unknown.In an effort to gain information about the function of A2, the gene for A2 was cloned into Escherichia coli, and the nucleotide sequence was determined. From the derived amino acid sequence, it was predicted that A2 consists largely of two ATP-binding domains. Component A2 shares homology with the "ATP-binding cassette" family of proteins, most of which are involved in energy-dependent transport systems. Component A2 was the first archaeal protein to be added to this family. In addition, it was demonstrated that functional A2 could be purified from E. coli.Components of the MR system were studied biochemically. Component A3a was purified according to previous protocols. However, the A3a fraction obtained often catalyzed CH$\sb4$ formation in the absence of added A2, MR, and ATP. Component A3a could not be separated from the CH$\sb4$-forming activity. Active MR, which does not require additional protein components or ATP for activity, was purified and shown to differ from inactive MR in that activity was not dependent upon light and was not inhibited by bathophenanthroline disulfonate. Both active MR and its nickel cofactor, F$\sb{430},$ catalyzed CH$\sb4$ formation when CH$\sb3$-S-CoM was replaced with CH$\sb3$-B$\sb .$In a separate study, the requirement for a heat-stable factor for growth of Methanomicrobium mobile was studied. Previously, it had been shown that boiled cell-free extract (BCE) from M. thermoautotrophicum replaced rumen fluid for growth of M. mobile. By measuring growth of M. mobile in the presence of known methanogenic cofactors, it was shown that 7-mercaptoheptanoylthreonine phosphate (HS-HTP) replaced BCE for growth. It was also shown that the growth requirement might be satisfied by 7-mercaptoheptanoic acid, a biosynthetic precursor of HS-HTP, plus a second, unidentified factor in BCE. Issue Date: 1993 Type: Text Description: 125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993. URI: http://hdl.handle.net/2142/72526 Other Identifier(s): (UMI)AAI9411677 Date Available in IDEALS: 2014-12-17 Date Deposited: 1993
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