Dissertation/Thesis Abstract

The Mass of L-Pyrrolysine in Methylamine Methyltransferases and the Role of its Imine Bond in Catalysis
by Soares, Jitesh Anthony Aloysius, Ph.D., The Ohio State University, 2008, 218; 10631272
Abstract (Summary)

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 NaBH 4 was found to inhibit their enzymatic activity. Mass spectrometry showed that L-pyrrolysine was the only detectable site of NaBH 4 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.

Indexing (document details)
Advisor: Krzycki, Joseph
Commitee: Daniels, Charles, Morrison, Mark, Tabita, F. Robert
School: The Ohio State University
Department: Microbiology
School Location: United States -- Ohio
Source: DAI-B 78/11(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Microbiology
Keywords: Atp-dependent redox-activation enzyme, Methylamine methyltransferase, Pyrrolysine, Pyrrolysine function, Pyrrolysine structure
Publication Number: 10631272
ISBN: 9780355015423
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