Poster abstracts

Poster number 59 submitted by Jonathan Picking

Characterization of MtgB, a pyrrolysine-lacking homolog of the pyrrolysine-containing trimethylamine-corrinoid methyltransferase

Jonathan W. Picking (The Ohio State Biochemistry Program, The Ohio State University), Joseph A. Krzycki (Microbiology, The Ohio State University)

Abstract:
Pyrrolysine (pyl), sometimes called the 22nd amino acid, seemingly exists solely to assist in the microbial transfer of a methyl group from methylated amines to a corrinoid protein (as in the first step of methanogenesis from methylamines). MttB, the trimethylamine methyltransferase, is a TIM barrel containing pyl in the putative active site cleft. The proposed mechanism for methyl transfer involves pyl acting as a “handle,” forming a covalent bond with trimethylamine, orienting it for attack by the highly nucleophilic Co(I) of the corresponding corrinoid protein, MttC.

Pyl may have come to this function by entering an existing family of methyltransferases. Indeed, we identified many MttB homologues that lack pyl. One such homolog, Desulfitobacterium hafniense MtgB, was subsequently shown to act as a glycine betaine-corrinoid methyltransferase. In silico analysis of MtgB revealed four conserved amino acid residues likely to participate in binding and catalysis. An arginine (R309) located within the barrel is likely to coordinate the carboxylate group of TMG. Two aromatic residues within the barrel but nearer to the surface (Y96 and F353) may stabilize the methylated nitrogen of TMG via cation-π interactions. A nearby histidine (H345) would then be well positioned to participate in catalysis. Such binding of TMG could orient the methyl group for attack by the Co(I) of a corrinoid protein, consistent with the conserved architecture of corrinoid-dependent methyltransferases.

To investigate this model, mutations were generated at each of the four positions. Preliminary in vitro methylation assays indicate that each mutation reduces the activity of the enzyme below the level of detection. Recently, MtgB was crystallized in the presence of TMG and the corrinoid cofactor. The forthcoming solution of the substrate-bound structure will further refine this model, ultimately providing insight into the larger family of methyltransferases that include pyl.

Keywords: Pyrrolysine, Methyltransferase, Corrinoid