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The Mass Of L-Pyrrolysine In Methylamine Methyltransferases And The Role Of Its Imine Bond In Catalysis

Soares, Jitesh A A
http://rave.ohiolink.edu/etdc/view?acc_num=osu1204576355

Abstract Details

2008, Doctor of Philosophy, Ohio State University, Microbiology.
Methanosarcina barkeri is an archaeon capable of producing methane from methylamines. Methylamine methyltransferases initiate methanogenesis from methylamines by transferring methyl groups to a cognate corrinoid protein. Each gene encoding a methylamine methyltransferase has been shown to contain a single in-frame amber codon. Further studies have shown that in the monomethylamine methyltransferase, mtmB, the amber codon encodes a novel amino acid, L-pyrrolysine. X-ray crystal structures of MtmB have shown that the structure of this amino acid is a lysine residue with the epsilon-nitrogen in amide linkage to a (4R, 5R)-4-substituted pyrrolyine-5-carboxylate ring. However, these structures did not allow an assignment of the pyrroline ring C4 substituent as a methyl or amine group. In this thesis (Chapter 2) mass spectrometry of chymotryptic digests of methylamine methyltransferases is employed to show that pyrrolysine in present in all three types of methylamine methyltransferase at the position corresponding to the amber codon in their respective genes. The mass of this amber-encoded residue was observed to coincide with the predicted mass of pyrrolysine with a methyl- group at the C4 position. The x-ray crystal structures showed that pyrrolysine had electrophilic character suggesting the presence of an imine bond which could play a role in catalysis. In Chapter 3, the role of this imine bond in catalysis is probed with NaBH4. Treatment of methylamine methyltransferases with NaBH4 was found to inhibit their enzymatic activity. Mass spectrometry showed that L-pyrrolysine was the only detectable site of NaBH4 reduction in these methylamine methyltransferases. These data were consistent with the hypothesis that L-pyrrolysine plays a role in catalysis. During methanogenesis from methylamines, methylamine methyltransferases methylate cognate corrinoid proteins. During catalysis, the corrinoids oscillate between a supernucleophilic Co(I)-form and the CH3-Co(III) state. When in the Co(I)-form, the cobalt center is prone to oxidative inactivation to the Co(II)-form in methyltransferases. In Chapter 4 of this dissertation, a novel ATP-dependent redox activator, RAM is shown to play a role in the direct reduction of these corrinoid proteins.
Joseph Krzycki, PhD (Advisor)
Charles Daniels, PhD (Committee Member)
Mark Morrison, PhD (Committee Member)
F. Robert Tabita, PhD (Committee Member)
218 p.
Microbiology
pyrrolysine; pyrrolysine structure; pyrrolysine function; ATP-dependent redox-activation enzyme; methylamine methyltransferase

Recommended Citations

Citations

  • Soares, J. A. A. (2008). The Mass Of L-Pyrrolysine In Methylamine Methyltransferases And The Role Of Its Imine Bond In Catalysis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1204576355

    APA Style (7th edition)

  • Soares, Jitesh. The Mass Of L-Pyrrolysine In Methylamine Methyltransferases And The Role Of Its Imine Bond In Catalysis. 2008. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1204576355.

    MLA Style (8th edition)

  • Soares, Jitesh. "The Mass Of L-Pyrrolysine In Methylamine Methyltransferases And The Role Of Its Imine Bond In Catalysis." Doctoral dissertation, Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1204576355

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.